Novel immunotherapy against several tumors including neuronal and brain tumors

ABSTRACT

The present invention relates to peptides, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated cytotoxic T cell (CTL) peptide epitopes, alone or in combination with other tumor-associated peptides that serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses. The present invention relates to 30 peptide sequences and their variants derived from HLA class I and class II molecules of human tumor cells that can be used in vaccine compositions for eliciting anti-tumor immune responses.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. application Ser. No.13/346,598, filed on Jan. 9, 2012, which is a divisional of U.S.application Ser. No. 12/571,776, filed on Oct. 1, 2009, which claimspriority to U.S. Provisional Application No. 61/105,928, filed on Oct.16, 2008, EP Application No. 08 017 305.7, filed on Oct. 1, 2008, and EPApplication No. 08 017 921.1, filed on Oct. 13, 2008, and InternationalApplication No. PCT/EP2009/006980 filed Sep. 28, 2009, each of which ishereby incorporated by reference in its entirety.

BACKGROUND

1. Field of the Invention

The present invention relates to peptides, nucleic acids and cells foruse in immunotherapeutic methods. In particular, the present inventionrelates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated cytotoxic T cell (CTL) peptideepitopes, alone or in combination with other tumor-associated peptidesthat serve as active pharmaceutical ingredients of vaccine compositionsthat stimulate anti-tumor immune responses. The present inventionrelates to 30 peptide sequences and their variants derived from HLAclass I and class II molecules of human tumor cells that can be used invaccine compositions for eliciting anti-tumor immune responses.

2. Description of Related Art

Gliomas are brain tumors originating from glial cells in the nervoussystem. Glial cells, commonly called neuroglia or simply glia, arenon-neuronal cells that provide support and nutrition, maintainhomeostasis, form myelin, and participate in signal transmission in thenervous system. The two most important subgroups of gliomas areastrocytomas and oligodendrogliomas, named according to the normal glialcell type from which they originate (astrocytes or oligodendrocytes,respectively). Belonging to the subgroup of astrocytomas, glioblastomamultiforme (referred to as glioblastoma hereinafter) is the most commonmalignant brain tumor in adults and accounts for approx. 40% of allmalignant brain tumors and approx. 50% of gliomas. It aggressivelyinvades the central nervous system and is ranked at the highestmalignancy level (grade IV) among all gliomas. Although there has beensteady progress in their treatment due to improvements in neuroimaging,microsurgery, diverse treatment options, such as temozolomide orradiation, glioblastomas remain incurable. The lethal rate of this braintumor is very high: the average life expectancy is 9 to 12 months afterfirst diagnosis. The 5-year survival rate during the observation periodfrom 1986 to 1990 was 8.0%. To date, the five-year survival ratefollowing aggressive therapy including gross tumor resection is stillless than 10%. Accordingly, there is a strong medical need for analternative and effective therapeutic method.

Tumor cells of glioblastomas are the most undifferentiated ones amongbrain tumors, so the tumor cells have high potential of migration andproliferation and are highly invasive, leading to very poor prognosis.Glioblastomas lead to death due to rapid, aggressive, and infiltrativegrowth in the brain. The infiltrative growth pattern is responsible forthe unresectable nature of these tumors. Glioblastomas are alsorelatively resistant to radiation and chemotherapy, and, therefore,post-treatment recurrence rates are high. In addition, the immuneresponse to the neoplastic cells is rather ineffective in completelyeradicating all neoplastic cells following resection and radiationtherapy.

Glioblastoma is classified into primary glioblastoma (de novo) andsecondary glioblastoma, depending on differences in the gene mechanismduring malignant transformation of undifferentiated astrocytes or glialprecursor cells. Secondary glioblastoma occurs in a younger populationof up to 45 years of age. During 4 to 5 years, on average, secondaryglioblastoma develops from lower-grade astrocytoma throughundifferentiated astrocytoma. In contrast, primary glioblastomapredominantly occurs in an older population with a mean age of 55 years.Generally, primary glioblastoma occurs as fulminant glioblastomacharacterized by tumor progression within 3 months from the state withno clinical or pathological abnormalities (Pathology and Genetics of theNervous Systems. 29-39 (IARC Press, Lyon, France, 2000)).

Glioblastoma migrates along myelinated nerves and spreads widely in thecentral nervous system. In most cases surgical treatment shows onlylimited sustainable therapeutic effect. Malignant glioma cells evadedetection by the host's immune system by producing immunosuppressiveagents that impair T cell proliferation and production of theimmune-stimulating cytokine IL-2. Intracranial neoplasms can arise fromany of the structures or cell types present in the CNS, including thebrain, meninges, pituitary gland, skull, and even residual embryonictissue. The overall annual incidence of primary brain tumors in theUnited States is 14 cases per 100,000. The most common primary braintumors are meningiomas, representing 27% of all primary brain tumors,and glioblastomas, representing 23% of all primary brain tumors (whereasglioblastomas account for 40% of malignant brain tumor in adults). Manyof these tumors are aggressive and of high grade. Primary brain tumorsare the most common solid tumors in children and the second mostfrequent cause of cancer death after leukemia in children.

The search for effective treatment of glioblastomas in patients is stillongoing today. Immunotherapy, or treatment via recruitment of the immunesystem, to fight these neoplastic cells has been investigated. Firstencouraging results with immuno-therapeutic approaches in patientssuffering from glioblastoma were obtained by Northwest Biotherapeuticsusing “DCVax Brain”, a cell-based vaccination approach employingpatient-derived dendritic cells loaded with autologous tumor celllysates, and by Celldex, which used a peptide from EGFRvIII for inducingantigen-specific CTL responses, which in turn correlated with prolongedmedian survival times compared to median survival times obtained whenusing standard treatment (Heimberger et al., 2006).

Colorectal Carcinoma

According to the American Cancer Society, colorectal cancer (CRC) is thethird most common cancer in the US, afflicting more than 175,000 newpatients each year. In the US, Japan, France, Germany, Italy, Spain andthe UK, it affects more than 480,000 patients. It is one of the mostcommon causes of cancer mortality in developed countries. The 1- and5-year relative survival for persons with colorectal cancer is 84% and64%, respectively. Survival continues to decline beyond 5 years to 57%at 10 years after diagnosis. When colorectal cancers are detected at anearly, localized stage, the 5-year survival is 90%; however, only 39% ofcolorectal cancers are diagnosed at this stage, mostly due to low ratesof screening. After the cancer has spread regionally to involve adjacentorgans or lymph nodes, the 5-year survival drops to 68%. For personswith distant metastases, 5-year survival is 10%.

Research suggests that the onset of colorectal cancer is the result ofinteractions between inherited and environmental factors. In most casesadenomatous polyps appear to be precursors to colorectal tumors; howeverthe transition may take many years. The primary risk factor forcolorectal cancer is age, with 90% of cases diagnosed over the age of 50years. Other risk factors for colorectal cancer according to theAmerican Cancer Society include alcohol consumption, a diet high in fatand/or red meat and an inadequate intake of fruits and vegetables.Incidence continues to rise, especially in areas such as Japan, wherethe adoption of westernized diets with excess fat and meat intake and adecrease in fiber intake may be to blame. However, incidence rates arerising not as fast as previously which may be due to increasingscreening and polyp removal, thus preventing progression of polyps tocancer.

As in most solid tumors, first line treatment is surgery, however, itsbenefits remain confined to early-stage patients, yet a significantproportion of patients are diagnosed in advanced stages of the disease.For advanced colorectal cancer chemotherapy regimens based onfluorouracil-based regimens are standard of care. The majority of theseregimens are the so-called FOLFOX (infusional 5-FU/leucovorin plusoxaliplatin) and FOLFIRI (irinotecan, leucovorin, bolus andcontinuous-infusion 5-FU) protocols.

The introduction of third-generation cytotoxics such as irinotecan andoxaliplatin has raised the hope of significantly improving efficacy, butprognosis is still relatively poor, and the survival rate generallyremains at approximately 20 months in metastatic disease and, as aresult, the unmet needs in the disease remain high.

Recently a novel generation of drugs, molecular-targeted agents, such asAvastin® (bevacizumab) and Erbitux® (cetuximab), became available andabout 40 compounds are in late-stage clinical development for differentstages of colorectal cancer. Combinations of several of these compoundsincrease the number of potential treatment options to be expected forthe future. The vast majority of substances are in phase 2, with theEGFR being addressed by these compounds more often than any other targetin colorectal cancer trials, which is due to the fact that in ˜80% ofpatients with colorectal cancer EGFR expression is upregulated.

Clinical trials with stage II patients combining chemotherapy with therecently approved monoclonal antibodies (mAbs) (cetuximab+irinotecan orFOLFOX4; bevacizumab as a single-agent or together with FOLFOX4) arecurrently being conducted. Three to four year observation periods areexpected for statistically significant results from these trials.

Monoclonal antibodies (mAbs) presently used in oncology in general havean excellent chance of not interfering with active immunotherapy. Infact, there is preclinical (GABRILOVICH 1999) and clinical evidencesuggesting that depletion of VEGF (by bevacizumab) contributespositively to DC-mediated activation of T-cells (Osada T, Chong G,Tansik R, Hong T, Spector N, Kumar R, Hurwitz H I, Dev I, Nixon A B,Lyerly H K, Clay T, Morse M A. The effect of anti-VEGF therapy onimmature myeloid cell and dendritic cells in cancer patients. CancerImmunol Immunother. 2008 Jan. 10.).

Prostate Carcinoma and Other Tumors

With an estimated 27,050 deaths in 2007, prostate cancer is a leadingcause of cancer death in men. Although death rates have been decliningamong white and African American men since the early 1990s, rates inAfrican American men remain more than twice as high as those in whitemen. Prostate cancer is the most frequently diagnosed cancer in men. Forreasons that remain unclear, incidence rates are significantly higher inAfrican American men than in white men. Incidence rates of prostatecancer have changed substantially over the last 20 years: rapidlyincreasing from 1988-1992, declining sharply from 1992-1995, andincreasing modestly since 1995. These trends in large part reflectincreased prostate cancer screening with the prostate-specific antigen(PSA) blood test. Moderate incidence increases in the last decade aremost likely attributable to widespread PSA screening among men youngerthan 65. Prostate cancer incidence rates have leveled off in men aged 65years and older. Rates peaked in white men in 1992 (237.6 per 100,000men) and in African American men in 1993 (342.8 per 100,000 men).

Treatment for prostate cancer may involve watchful waiting, surgery,radiation therapy, High Intensity Focused Ultrasound (HIFU),chemotherapy, cryosurgery, hormonal therapy, or some combination. Whichoption is best depends on the stage of the disease, the Gleason score,and the PSA level. Other important factors are the man's age, hisgeneral health, and his feelings about potential treatments and theirpossible side effects. Because all treatments can have significant sideeffects, such as erectile dysfunction and urinary incontinence,treatment discussions often focus on balancing the goals of therapy withthe risks of lifestyle alterations.

If the cancer has spread beyond the prostate, treatment optionssignificantly change, so most doctors who treat prostate cancer use avariety of nomograms to predict the probability of spread. Treatment bywatchful waiting, HIFU, radiation therapy, cryosurgery, and surgery aregenerally offered to men whose cancer remains within the prostate.Hormonal therapy and chemotherapy are often reserved for disease whichhas spread beyond the prostate. However, there are exceptions: radiationtherapy may be used for some advanced tumors, and hormonal therapy isused for some early stage tumors. Cryotherapy, hormonal therapy, andchemotherapy may also be offered if initial treatment fails and thecancer progresses.

In a significant number of patients with prostate carcinoma who undergoradical prostatectomy because of clinically suspected organ-limitedgrowth, a definitive histological workup of the surgical preparationshows a locally extensive tumor extending beyond the borders of theorgan. These patients have a high risk for early local recurrence,usually detectable as an increasing PSA level in terms of a biochemicalrelapse. Therapeutic options in this situation include externalradiotherapy and hormone ablation; however, the value of thesetherapeutic approaches, especially with respect to prolonging thepatient's long-term survival, must not be regarded as proven. Inaddition, possible treatment-associated complications such as thedevelopment of urethral strictures (radiotherapy), loss of libido andimpotence, the risk of a reduction in skeletal calcium salts in terms ofosteoporosis, and a markedly increased risk of pathologic bone fractures(hormone ablation) must be considered.

More than 90% of all prostate cancers are discovered in the local andregional stages; the 5-year relative survival rate for patients whosetumors are diagnosed at these stages approaches 100%. Over the past 25years, the 5-year survival rate for all stages combined has increasedfrom 69% to nearly 90%. According to the most recent data, relative10-year survival is 93% and 15-year survival is 77%. The dramaticimprovements in survival, particularly at 5 years, are partlyattributable to earlier diagnosis and improvements in treatment.Nevertheless, the survival rate drops significantly after the spreadingto other tissues and organs.

Lung Cancer

Estimated 210,000 new cases are expected in 2007 in the USA, accountingfor about 15% of cancer diagnoses. The incidence rate is decliningsignificantly in men, from a high of 102 cases per 100,000 in 1984 to78.5 in 2003. In women, the rate is approaching a plateau after a longperiod of increase. Lung cancer is classified clinically as small cell(13%) or non-small cell (87%) for the purposes of treatment.

Lung cancer accounts for the most cancer-related deaths in both men andwomen. An estimated 160,390 deaths, accounting for about 29% of allcancer deaths, are expected to occur in 2007. Since 1987, more womenhave died each year from lung cancer than from breast cancer. Deathrates have continued to decline significantly in men from 1991-2003 byabout 1.9% per year. Female lung cancer death rates are approaching aplateau after continuously increasing for several decades. These trendsin lung cancer mortality reflect the decrease in smoking rates over thepast 30 years.

Treatment options are determined by the type (small cell or non-smallcell) and stage of cancer and include surgery, radiation therapy,chemotherapy, and targeted biological therapies such as bevacizumab(Avastin®) and erlotinib (Tarceva®). For localized cancers, surgery isusually the treatment of choice. Recent studies indicate that survivalwith early-stage, non-small cell lung cancer is improved by chemotherapyfollowing surgery. Because the disease has usually spread by the time itis discovered, radiation therapy and chemotherapy are often used,sometimes in combination with surgery. Chemotherapy alone or combinedwith radiation is the usual treatment of choice for small cell lungcancer; on this regimen, a large percentage of patients experienceremission, which is long lasting in some cases.

The 1-year relative survival for lung cancer has slightly increased from37% in 1975-1979 to 42% in 2002, largely due to improvements in surgicaltechniques and combined therapies. However, the 5-year survival rate forall stages combined is only 16%. The survival rate is 49% for casesdetected when the disease is still localized; however, only 16% of lungcancers are diagnosed at this early stage.

There thus remains a need for new efficacious and safe treatment optionfor glioblastoma, prostate tumor, breast cancer, esophageal cancer,colorectal cancer, clear cell renal cell carcinoma, lung cancer, CNS,ovarian, melanoma, pancreatic cancer, squamous cell carcinoma, leukemiaand medulloblastoma and other tumors which show an overexpression ofsurvivin and/or the other proteins of the present invention, enhancingthe well-being of the patients without using chemotherapeutic agents orother agents which may lead to severe side effects.

SUMMARY OF THE INVENTION

In a first aspect thereof, the present invention relates to a peptidecomprising a sequence selected from the group of SEQ ID NO:1 to SEQ IDNO:30, or a variant thereof that is at least 85% homologous to SEQ IDNO:1 to SEQ ID NO:30, or a variant thereof that induces T cellscross-reacting with said variant peptide; wherein said peptide is notthe full-length polypeptide of human survivin. Preferably, said peptideis selected from a peptide having a specific HLA-subtype, such asHLA-A*02 or HLA-DR.

In a second aspect thereof, the present invention relates to a nucleicacid, encoding a peptide according to the present invention or anexpression vector capable of expressing said nucleic acid.

In a third aspect thereof, the present invention relates to a host cellcomprising the nucleic acid or the expression vector according to thepresent invention, wherein said host cell preferably is an antigenpresenting cell, in particular a dendritic cell or antigen presentingcell.

In a fourth aspect thereof, the present invention relates to an in vitromethod for producing activated cytotoxic T lymphocytes (CTL), comprisingcontacting in vitro CTL with antigen loaded human class I MHC moleculesexpressed on the surface of a suitable antigen-presenting cell or anartificial construct mimicking an antigen-presenting cell for a periodof time sufficient to activate said CTL in an antigen specific manner,wherein said antigen is a peptide according to the present invention.

In a fifth aspect thereof, the present invention relates to the use of apeptide according to the present invention, the nucleic acid or theexpression vector according to the present invention, the cell accordingto the present invention, or an activated cytotoxic T lymphocyteproduced according to the present invention for the treatment of canceror for the manufacture of a medicament against cancer, wherein saidmedicament preferably is a vaccine. Preferably, said cancer is selectedfrom astrocytoma, pilocytic astrocytoma, dysembryoplasticneuroepithelial tumor, oligodendrogliomas, ependymoma, glioblastomamultiforme, mixed gliomas, oligoastrocytomas, medulloblastoma,retinoblastoma, neuroblastoma, germinoma, teratoma, gangliogliomas,gangliocytoma, central gangliocytoma, primitive neuroectodermal tumors(PNET, e.g. medulloblastoma, medulloepithelioma, neuroblastoma,retinoblastoma, ependymoblastoma), tumors of the pineal parenchyma (e.g.pineocytoma, pineoblastoma), ependymal cell tumors, choroid plexustumors, neuroepithelial tumors of uncertain origin (e.g. gliomatosiscerebri, astroblastoma), glioblastoma prostate tumor, breast cancer,esophageal cancer, colon cancer, colorectal cancer, renal cellcarcinoma, clear cell renal cell carcinoma, lung cancer, CNS, ovarian,melanoma pancreatic cancer, squamous cell carcinoma, leukemia andmedulloblastoma, and other tumors or cancers showing an overexpressionof Survivin and/or the other proteins of the present invention.

In a sixth aspect thereof, the present invention relates to a kit,comprising: (a) a container that contains a pharmaceutical compositioncontaining a peptide according to the present invention, the nucleicacid or the expression vector according to the present invention, a cellaccording to the present invention, or an activated cytotoxic Tlymphocyte according to the present invention, in solution or inlyophilized form; (b) optionally, a second container containing adiluent or reconstituting solution for the lyophilized formulation; (c)optionally, at least one peptide selected from the group consisting ofthe peptides according to SEQ ID NOs 1 to 30, and (d) optionally,instructions for the use of the solution and/or the reconstitutionand/or use of the lyophilized formulation. In a preferred embodiment thepeptide is selected from the group of SEQ ID NOs 1 to SEQ ID:24.

In a seventh aspect thereof, the present invention relates to a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically bindable to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

In an eighth aspect thereof, the present invention relates to anantibody that specifically binds to a human major histocompatibilitycomplex (MHC) class I or II being complexed with a HLA-restrictedantigen, wherein the antibody preferably is a polyclonal antibody,monoclonal antibody and/or a chimeric antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D demonstrate the ESI-liquid chromatography mass spectraidentifying tumor associated peptides (TUMAPs) IGF2BP3-001 fromglioblastoma sample GB6010 that was presented in a MHC class Irestricted manner.

FIGS. 2A-2D depict the mRNA expression profile of the target genes ofthe invention that are highly-overexpressed in glioblastoma samples.Expression of these genes is absent or very low in normal tissues whileit is strongly increased in glioblastoma samples. Relative mRNAexpressions are shown for several normal tissues and individualglioblastoma multiforme (GBM) samples measured by gene chip analysis.Values are relative to expression levels on normal kidney (value alwaysarbitrarily set to 1.0). Values for normal tissues were generated withcommercially available mRNA pools. Letters in brackets indicate the“detection call” as given by the analysis software. The “detection call”designates whether a transcript was specifically detected in the sampleat all or whether no significant detection could be observed. It cantake the values “P” (present), “A” (absent), or “M” (marginallydetected).

FIG. 3 shows the tetramer analysis of microsphere driven proliferationof CSP-001 and NLGN4X-001 specific CD8+ lymphocytes from peripheralblood of a healthy donor. 1×10⁶ CD8+ enriched PBMCs per well werestimulated weekly with microspheres coupled to anti-CD28 plus highdensity tumor antigen A*0201/CSP-001 (left panel) or anti-CD28 plus highdensity tumor antigen A*0201/NLGN4X-001 (right panel). After threestimulations in vitro, all cells were stained with antibody CD8 FITC,and fluorescently-labeled tetramers A*0201/CSP-001 andA*0201/NLGN4X-001. Cells are gated on CD8+ lymphocytes; numbersrepresent percentage of cells in the indicated quadrant among CD8+lymphocytes.

FIG. 4 shows the affinity of HLA class I peptides of the invention tothe MHC molecule coded by the HLA-A*0201 allele. Dissociation constants(K_(D)) of HLA class I TUMAPs from the invention and the control peptideHBV-001 (strong A*02 binder) were measured by an ELISA-based MHCrefolding assay.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are typically 9 amino acids in length, but can be as short as 8amino acids in length, and as long as 16 or 10, 11, 12, 13, 14 or 15amino acids in length.

The term “oligopeptide” is used herein to designate a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.The length of the oligopeptide is not critical to the invention, as longas the correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 14 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein, or polynucleotide coding for such amolecule is “immunogenic” (and thus an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse.

A T cell “epitope” requires a short peptide that is bound to a class Ior II MHC receptor, forming a ternary complex (MHC class I alpha chain,beta-2-microglobulin, and peptide) that can be recognized by a T cellbearing a matching T-cell receptor binding to the MHC/peptide complexwith appropriate affinity. Peptides binding to MHC class I molecules aretypically 8-14 amino acids in length, and most typically 9 amino acidsin length. T cell epitopes that bind to MHC class II molecules aretypically 12-30 amino acids in length. In the case of peptides that bindto MHC class II molecules, the same peptide and the corresponding T cellepitope may share a common core segment, but differ in the overalllength due to flanking sequences of differing lengths upstream of theamino-terminus of the core sequence and downstream of itscarboxy-terminus, respectively. MHC class II receptors have a more openconformation, peptides bound to MHC class II receptors arecorrespondingly not completely buried in the structure of the MHC classII molecule peptide-binding cleft as they are in the MHC class Imolecule peptide-binding cleft. Surprisingly this is not the case forthe peptide according to SEQ ID NO:1, as small variations in the lengthof the peptide lead to an extreme decrease of activity (see below).

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-A*11 are examples of different MHC class I alleles that can beexpressed from these loci.

There are three different loci in the human genome for MHC class IIgenes: HLA-DR, HLA-DQ, and HLA-DP. MHC class II receptors areheterodimers consisting of an alpha and a beta chain, both anchoring inthe cell membrane via a transmembrane region. HLA-DRB1*04, andHLA-DRB1*07 are two examples of different MHC class II beta alleles thatare known to be encoded in these loci. Class II alleles are verypolymorphic, e.g. several hundred different HLA-DRB1 alleles have beendescribed. Therefore, for therapeutic and diagnostic purposes a peptidethat binds with appropriate affinity to several different HLA class IIreceptors is highly desirable. A peptide binding to several differentHLA class II molecules is called a promiscuous binder.

As used herein, reference to a DNA sequence includes both singlestranded and double stranded DNA. Thus, the specific sequence, unlessthe context indicates otherwise, refers to the single strand DNA of suchsequence, the duplex of such sequence with its complement (doublestranded DNA) and the complement of such sequence. The term “codingregion” refers to that portion of a gene which either naturally ornormally codes for the expression product of that gene in its naturalgenomic environment, i.e., the region coding in vivo for the nativeexpression product of the gene.

The coding region can be from a normal, mutated or altered gene, or caneven be from a DNA sequence, or gene, wholly synthesized in thelaboratory using methods well known to those of skill in the art of DNAsynthesis.

The term “nucleotide sequence” refers to a heteropolymer ofdeoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment,” when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnontranslated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “open reading frame (ORF)” means a series of triplets codingfor amino acids without any termination codons and is a sequence(potentially) translatable into protein.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, the claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly contemplated.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form.” As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform.

The term “active fragment” means a fragment that generates an immuneresponse (i.e., has immunogenic activity) when administered, alone oroptionally with a suitable adjuvant, to an animal, such as a mammal, forexample, a rabbit or a mouse, and also including a human, such immuneresponse taking the form of stimulating a T-cell response within therecipient animal, such as a human. Alternatively, the “active fragment”may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion,” “segment,” and “fragment,” whenused in relation to polypeptides, refer to a continuous sequence ofresidues, such as amino acid residues, which sequence forms a subset ofa larger sequence. For example, if a polypeptide were subjected totreatment with any of the common endopeptidases, such as trypsin orchymotrypsin, the oligopeptides resulting from such treatment wouldrepresent portions, segments or fragments of the starting polypeptide.This means that any such fragment will necessarily contain as part ofits amino acid sequence a segment, fragment or portion, that issubstantially identical, if not exactly identical, to a sequence of SEQID NO: 1 to 30, which correspond to the naturally occurring, or “parent”proteins of the SEQ ID NO: 1 to 30. When used in relation topolynucleotides, such terms refer to the products produced by treatmentof said polynucleotides with any of the common endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical,” when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The Percent Identity isthen determined according to the following formula:

Percent Identity=100[I−(C/R)]

-   -   wherein C is the number of differences between the Reference        Sequence and the Compared Sequence over the length of alignment        between the Reference Sequence and the Compared Sequence,        wherein    -   (i) each base or amino acid in the Reference Sequence that does        not have a corresponding aligned base or amino acid in the        Compared Sequence and    -   (ii) each gap in the Reference Sequence and    -   (iii) each aligned base or amino acid in the Reference Sequence        that is different from an aligned base or amino acid in the        Compared Sequence, constitutes a difference;    -   and R is the number of bases or amino acids in the Reference        Sequence over the length of the alignment with the Compared        Sequence with any gap created in the Reference Sequence also        being counted as a base or amino acid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified PercentIdentity.

The original peptides disclosed herein can be modified by thesubstitution of one or more residues at different, possibly selective,sites within the peptide chain, if not otherwise stated. Suchsubstitutions may be of a conservative nature, for example, where oneamino acid is replaced by an amino acid of similar structure andcharacteristics, such as where a hydrophobic amino acid is replaced byanother hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,amino acids possessing non-standard R groups (i.e., R groups other thanthose found in the common 20 amino acids of natural proteins) may alsobe used for substitution purposes to produce immunogens and immunogenicpolypeptides according to the present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would simultaneously be substituted.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted CTLs, effector functions may be lysisof peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation. For MHC class II-restricted T helper cells,effector functions may be peptide induced secretion of cytokines,preferably, IFN-gamma, TNF-alpha, IL-4, IL5, IL-10, or IL-2, orpeptide-induced degranulation. Possible effector functions for CTLs andT helper cells are not limited to this list.

Preferably, when the CTLs specific for a peptide of SEQ IDs NO: 1 to 30are tested against the substituted peptides, the peptide concentrationat which the substituted peptides achieve half the maximal increase inlysis relative to background is no more than about 1 mM, preferably nomore than about 1 μM, more preferably no more than about 1 nM, and stillmore preferably no more than about 100 pM, and most preferably no morethan about 10 pM. It is also preferred that the substituted peptide berecognized by CTLs from more than one individual, at least two, and morepreferably three individuals.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than 4 residues from thereference peptide, as long as they have substantially identicalantigenic activity.

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has now raised thepossibility of using a host's immune system to foster an immune responsethat is specific for target antigens expressed on the surface of tumorcells and which through this mechanism of action is capable of inducingregression, stasis or slowed-down growth of the tumor. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofcytotoxic T cells (CTL) from tumor-infiltrating cell populations or fromperipheral blood suggests that such cells play an important role innatural immune defenses against cancer (Cheever et al., 1993; Zeh, IIIet al., 1999). Based on the analysis of 415 specimens from patientssuffering from colorectal cancer, Galon et al. were able to demonstratethat type, density and location of immune cells in tumor tissue areactually a better predictor for survival of patients than the widelyemployed TNM-staging of tumors (Galon et al., 2006).

MHC class I present peptides that result from proteolytic cleavage ofpredominantly endogenous proteins, DRIPs and larger peptides. MHC classII molecules can be found predominantly on professional antigenpresenting cells (APCs), and primarily present peptides of exogenous ortransmembrane proteins that are taken up by APCs during the course ofendocytosis, and are subsequently processed (Cresswell, 1994). Complexesof peptide and MHC class I molecules are recognized by CD8-positivecytotoxic T-lymphocytes bearing the appropriate TCR (T-cell receptor),and complexes of peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells (Wangand Livingstone, 2003; Sun and Bevan, 2003; Shedlock and Shen, 2003).Initially, the priming and expansion of CTLs in lymph nodes is supportedby CD4+ T-cells (Schoenberger et al., 1998). One mechanism thereforemight be the guidance of naive CD8+ cells to the place of functionalCD4+ T-cell—APC interaction (Castellino et al., 2006). Finally, thegeneration of functional CD8+ memory cells is in most cases dependent onCD4+ T-cell assistance (Sun and Bevan, 2003; Janssen et al., 2003). Forthese reasons, the identification of CD4-positive T-cell epitopesderived from tumor associated antigens (TAA) is of great importance forthe development of pharmaceutical products for triggering anti-tumorimmune responses (Kobayashi et al., 2002; Qin et al., 2003; Gnjatic etal., 2003). At the tumor site, T helper cells, support a CTL friendlycytokine milieu (Qin and Blankenstein, 2000; Mortara et al., 2006) andattract effector cells, e.g. CTLS, NK cells, macrophages, granulocytes(Marzo et al., 2000; Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave surprisingly been found to express MHC class II molecules (Dengjelet al., 2006).

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CTL effector cells (i.e., CD8-positive T lymphocytes),CD4-positive T cells are sufficient for inhibiting manifestation oftumors via inhibition of angiogenesis by secretion of interferon-gamma(IFNy) (Qin and Blankenstein, 2000). Also the direct killing of tumorcells by cytotoxic CD4+ T cells via lymphotoxins and granzyme B has beenproposed (Penna et al., 1992; Littaua et al., 1992).

Additionally, it was shown that CD4-positive T cells recognizingpeptides from tumor-associated antigens presented by HLA class IImolecules can counteract tumor progression via the induction of antibody(Ab) responses (Kennedy et al., 2003).

In contrast to tumor-associated peptides binding to HLA class Imolecules, only a small number of class II ligands of tumor associatedantigens (TAA) have been described to date.

Since the constitutive expression of HLA class II molecules is usuallylimited to cells of the immune system (Mach et al., 1996), thepossibility of isolating class II peptides directly from primary tumorswas not considered possible. However, Dengjel et al. were recentlysuccessful in identifying a number of MHC Class II epitopes directlyfrom tumors (WO 2007/028574, EP 1 760 088 B1; (Dengjel et al., 2006).

The antigens that are recognized by the tumor specific cytotoxic Tlymphocytes, that is, their epitopes, can be molecules derived from allprotein classes, such as enzymes, receptors, transcription factors, etc.which are expressed and, as compared to unaltered cells of the sameorigin, up-regulated in cells of the respective tumor.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups (Novellino et al., 2005):

1. Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells (van der Bruggen et al., 1991) belong to thisclass, which was originally called cancer-testis (CT) antigens becauseof the expression of its members in histologically different humantumors and, among normal tissues, only in spermatocytes/spermatogonia oftestis and, occasionally, in placenta. Since the cells of testis do notexpress class I and II HLA molecules, these antigens cannot berecognized by T cells in normal tissues and can therefore be consideredas immunologically tumor-specific. Well-known examples for CT antigensare the MAGE family members or NY-ESO-1.

2. Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose; most are found inmelanomas and normal melanocytes. Many of these melanocytelineage-related proteins are involved in the biosynthesis of melanin andare therefore not tumor specific but nevertheless are widely used forcancer immunotherapy. Examples include, but are not limited to,tyrosinase and Melan-A/MART-1 for melanoma or PSA for prostate cancer.

3. Overexpressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir overexpression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, Survivin, Telomerase or WT1.

4. Tumor specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors.

5. TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins that are neither specific nor overexpressedin tumors but nevertheless become tumor associated by posttranslationalprocesses primarily active in tumors. Examples for this class arise fromaltered glycosylation patterns leading to novel epitopes in tumors asfor MUC1 or events like protein splicing during degradation, which mayor may not be tumor specific (Hanada et al., 2004; Vigneron et al.,2004).

6. Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

For proteins to be recognized by cytotoxic T-lymphocytes astumor-specific or -associated antigens, and in order to be used in atherapy, particular prerequisites must be fulfilled. The antigen shouldbe expressed mainly by tumor cells and not or in comparably smallamounts by normal healthy tissues. It is furthermore desirable, that therespective antigen is not only present in a type of tumor, but also inhigh concentrations (i.e. copy numbers of the respective peptide percell). Tumor-specific and tumor-associated antigens are often derivedfrom proteins directly involved in transformation of a normal cell to atumor cell due to a function e.g. in cell cycle control or suppressionof apoptosis. Additionally, also downstream targets of the proteinsdirectly causative for a transformation may be upregulated and thus maybe indirectly tumor-associated. Such indirectly tumor-associatedantigens may also be targets of a vaccination approach (Singh-Jasuja etal., 2004). In both cases it is essential that epitopes are present inthe amino acid sequence of the antigen, since such a peptide(“immunogenic peptide”) that is derived from a tumor associated antigenshould lead to an in vitro or in vivo T-cell-response.

Basically, any peptide able to bind a MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell with a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a tumorvaccine. The methods for identifying and characterizing the TAAs arebased on the use of CTL that can be isolated from patients or healthysubjects, or they are based on the generation of differentialtranscription profiles or differential peptide expression patternsbetween tumors and normal tissues (Lemmel et al., 2004; Weinschenk etal., 2002).

However, the identification of genes over-expressed in tumor tissues orhuman tumor cell lines, or selectively expressed in such tissues or celllines, does not provide precise information as to the use of theantigens being transcribed from these genes in an immune therapy. Thisis because only an individual subpopulation of epitopes of theseantigens are suitable for such an application since a T cell with acorresponding TCR has to be present and immunological tolerance for thisparticular epitope needs to be absent or minimal. It is thereforeimportant to select only those peptides from over-expressed orselectively expressed proteins that are presented in connection with MHCmolecules against which a functional T cell can be found. Such afunctional T cell is defined as a T cell that upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

T-helper cells play an important role in orchestrating the effectorfunction of CTLs in anti-tumor immunity. T-helper cell epitopes thattrigger a T-helper cell response of the T_(H1) type support effectorfunctions of CD8-positive killer T cells, which include cytotoxicfunctions directed against tumor cells displaying tumor-associatedpeptide/MHC complexes on their cell surfaces. In this waytumor-associated T-helper cell peptide epitopes, alone or in combinationwith other tumor-associated peptides, can serve as active pharmaceuticalingredients of vaccine compositions that stimulate anti-tumor immuneresponses.

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+CTLs (ligand: MHC class I molecule+peptide epitope) or by CD4-positiveT-helper cells (ligand: MHC class II molecule+peptide epitope) isimportant in the development of tumor vaccines.

Considering the severe side-effects and expense associated with treatingcancer better prognosis and diagnostic methods are desperately needed.Therefore, there is a need to identify other factors representingbiomarkers for cancer in general and glioblastoma in particular.Furthermore, there is a need to identify factors that can be used in thetreatment of cancer in general and glioblastoma in particular,

Furthermore there is no established therapeutic design for prostatecancer patients with biochemical relapse after radical prostatectomy,usually caused by residual tumor left in situ in the presence of locallyadvanced tumor growth. New therapeutic approaches that confer lowermorbidity with comparable therapeutic efficacy relative to the currentlyavailable therapeutic approaches would be desirable.

The present invention provides peptides that are useful in treatingglioblastoma, prostate cancer and other tumors that overexpress survivinand/or CSP and/or other peptides of the invention. These peptides werepartly directly shown by mass spectrometry to be naturally presented byHLA molecules on primary human glioblastoma samples (see example 1 andFIG. 1), or in the case of SEQ ID NO:26 predicted according to theSYFPEITHI prediction algorithm (Rammensee et al., 1995) to bepromiscuous binders to the HLA-DR alleles HLA-DRB1*01, DRB1*03, DRB1*04,DRB1*11, and DRB1*15. Based on this data and the frequencies of thesefrequent DRB1 alleles, it can be assumed that 92% of A*02-positiveCaucasians express at least one DRB1 allele that binds the peptideaccording to SEQ ID NO:26.

The source gene from which SEQ ID NO: 26 to 30 are derived—survivin—wasshown to be highly overexpressed in glioblastoma, prostate tumor, breastcancer, esophageal cancer, colorectal cancer, clear cell renal cellcarcinoma, lung cancer, CNS, ovarian, melanoma (Tamm et al. 1998)pancreatic cancer, squamous cell carcinoma, leukemia and medulloblastomacompared with normal tissues (see example 2 and FIG. 2) demonstrating ahigh degree of tumor association of the peptide, i.e. these peptides arestrongly presented on tumor tissue but not on normal tissues. WO2004/067023 describes MHC Class I-restricted peptides derived from thetumor associated antigen survivin, which peptides are capable of bindingto Class I HLA molecules at a high affinity.

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes/T cells. T cells can destroy the cells presenting therecognized HLA/peptide complex, e.g. glioblastoma tumor cells presentingthe derived peptides. T helper cells activated by the survivin-derivedpeptides can inhibit tumor vascularization, can attract effector cellsof the immune system and facilitate CTL priming, proliferation, and asustained CD8+ T-cell response.

All peptides of the present invention have been shown to be capable ofstimulating T cell responses (see Example 3 and FIG. 3). Thus, thepeptides are useful for generating an immune response in a patient bywhich tumor cells can be destroyed. An immune response in a patient canbe induced by direct administration of the described peptides orsuitable precursor substances (e.g. elongated peptides, proteins, ornucleic acids encoding these peptides) to the patient, ideally incombination with an agent enhancing the immunogenicity (i.e. anadjuvant). The immune response originating from such a therapeuticvaccination can be expected to be highly specific against tumor cellsbecause the target peptides of the present invention are not presentedon normal tissues in comparable copy numbers, preventing the risk ofundesired autoimmune reactions against normal cells in the patient.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt.

As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates) or hydrochloricacid (chlorides).

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from glioblastoma and since it was determined that thesepeptides are not present in normal tissues, these peptides can be usedto diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies can assist apathologist in diagnosis of cancer. Detection of certain peptides bymeans of antibodies, mass spectrometry or other methods known in the artcan tell the pathologist that the tissue is malignant or inflamed orgenerally diseased. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmunosurveillance. Thus, presence of peptides shows that this mechanismis not exploited by the analyzed cells.

The peptides might be used to analyze lymphocyte responses against thosepeptides such as T cell responses or antibody responses against thepeptide or the peptide complexed to MHC molecules. These lymphocyteresponses can be used as prognostic markers for decision on furthertherapy steps. These responses can also be used as surrogate markers inimmunotherapy approaches aiming to induce lymphocyte responses bydifferent means, e.g. vaccination of protein, nucleic acids, autologousmaterials, adoptive transfer of lymphocytes. In gene therapy settings,lymphocyte responses against peptides can be considered in theassessment of side effects. Monitoring of lymphocyte responses mightalso be a valuable tool for follow-up examinations of transplantationtherapies, e.g. for the detection of graft versus host and host versusgraft diseases.

The peptides can be used to generate and develop specific antibodiesagainst MHC/peptide complexes. These can be used for therapy, targetingtoxins or radioactive substances to the diseased tissue. Another use ofthese antibodies can be targeting radionuclides to the diseased tissuefor imaging purposes such as PET. This use can help to detect smallmetastases or to determine the size and precise localization of diseasedtissues.

In addition, they can be used to verify a pathologist's diagnosis of acancer based on a biopsied sample.

Table 1 shows the peptides according to the present invention, theirrespective SEQ ID NO:, the HLA alleles to which the respective peptidesbind, and the source proteins from which these peptides may arise. Ofspecial interest is the fact that the peptide according to SEQ ID NO:1binds to HLA-DR as well as HLA-A*02 thus eliciting two differentresponses.

TABLE 1 Peptides of the present invention SEQ ID Peptide HLA Source NO:Code Sequence Alleles Protein(s)  1 NLGN4X- NLDTLMTYV HLA-A*02 NLGN4X001  2 SLCO1C1- YLIAGIISL HLA-A*02 SLCO1C1 100  3 ACS-001 KIMERIQEVHLA-A*02 ACSBG1  4 BCA-001 FLGDPPEKL HLA-A*02 BCAN  5 BCA-002 ALWAWPSELHLA-A*02 BCAN  6 CHI3L1- TLYGMLNTL HLA-A*02 CHI3L1 010  7 CLIP2-SLNELRVLL HLA-A*02 CLIP2 001  8 DTNA-001 KLQDEAYQV HLA-A*02 DTNA  9EGFR-007 ALAVLSNYDA HLA-A*02 EGFR 10 FABP7- LTFGDVVAV HLA-A*02 FABP7 00111 GFAP-001 NLAQDLATV HLA-A*02 GFAP 12 GPR56- FLLSEPVAL HLA-A*02 GPR56002 13 GRI-001 NILEQIVSV HLA-A*02 GRIA4 14 IGF2BP3- KIQEILTQV HLA-A*02IGF2BP3 001 15 MLC-001 SVVEVIAGI HLA-A*02 MLC1 16 NES-001 GLQSQIAQVHLA-A*02 NES 17 NES-002 SLQENLESL HLA-A*02 NES 18 NES-003 FLFPGTENQHLA-A*02 NES EL 19 NES-004 NLAEELEGV HLA-A*02 NES 20 NR2E1- KIISEIQALHLA-A*02 NR2E1 001 21 NRCAM- GLWHHQTEV HLA-A*02 NRCAM 001 22 PDPN-001TLVGIIVGV HLA-A*02 PDPN 23 TNC-001 AMTQLLAGV HLA-A*02 TNC 24 TNC-002QLLAGVFLA HLA-A*02 TNC 25 CSP-001 TMLARLASA HLA-A*02 CSPG4 26 BIR-002TLGEFLKLD HLA-DR and BIRC5/ RERAKN HLA-A*02 Survivin 27 BIR-002aTLGEFLKLD HLA-DR BIRC5/ RERAKD Survivin 28 BIR-002b FTELTLGEF HLA-A1BIRC5/ Survivin 29 BIR-002c LMLGEFLKL HLA-A2 BIRC5/ Survivin 30 BIR-002dEPDLAQCFY HLA-B35 BIRC5/ Survivin

Chondroitin sulfate proteoglycan 4 (CSPG4)

CSPG4 (chondroitin sulfate proteoglycan) represents an integral membranechondroitin sulfate proteoglycan on nascent pericytes with a functionalrole in neovascularization (Ozerdem, 2006). During embryogenesis, theCSPG4 proteoglycan is expressed on immature capillary vessels, but asthe vessels mature they lose this expression. It is known as an earlycell surface melanoma progression marker implicated in stimulating tumorcell proliferation, migration and invasion. CSPG4 is strongly expressedon >90% of human melanoma lesions. Although CSPG4 is not strictly tumorspecific, tumor-reactive CD4+ T-cell responses in melanoma patients andhealthy individuals recognize CSPG₄₆₉₃₋₇₀₉ on HLA-DR11-expressingmelanoma cells in the absence of autoimmunity (Erfurt et al., 2007).

Expression of CSPG4 enhances integrin-mediated cell spreading, FAK(focal adhesion kinase) phosphorylation, and activation of ERK1/2(extracellular signal-regulated kinase) (Yang et al., 2004).Furthermore, there is accumulating evidence from in vitro data thatCSPG4 plays an important role in tumor angiogenesis. Thus,CSPG4-positive tumors have been found to have significantly increasedneovascularization rates and vascular volumes, and CSPG4 has been shownto sequester angiostatin, which normally inhibits endothelial cellproliferation and angiogenesis. Immature vessels also containCSPG4-positive pericytes, suggesting a role for this cell population inmodulating endothelial cell proliferation by blocking the inhibitoryeffects of angiostatin during vessel development (Chekenya et al.,2002b).

CSPG4 expression has also been described in some normal tissues besidesactivated pericytes such as endothelial cells, chondrocytes, smoothmuscle cells, certain basal keratinocytes within the epidermis, as wellas cells within the hair follicle (Campoli et al., 2004).

During angiogenesis and in response to CNS pathologies, the highlymotile CSPG4 cells undergo rapid morphological changes and are recruitedto sites where vessel growth and repair are occurring. CSPG4 isover-expressed by both tumor cells and pericytes on the blood vessels ofmalignant brain tumors (Chekenya and Pilkington, 2002). By implantingcells from an CSPG4-positive human glioma cell line into immunodeficientnude rat brains it was shown that these tumors had a highermicrovascular density in comparison to controls implying that CSPG4expression regulates both the function and the structure of thehost-derived tumor vasculature (Brekke et al., 2006). In a xenograftexperiment of implantation of GBM biopsy spheroids into nude rats, CSPG4was identified to be mainly associated with blood vessels on both thepericyte and basement membrane components of the tumor vasculature andthe expression was also associated with areas of high cellularproliferation (Chekenya et al., 2002a). Furthermore, CSPG4 expressionparalleled progression of the tumor in a glioma implantation model(Wiranowska et al., 2006). Malignant progression is maintained bycross-talk between the tumor and its stroma, where the activated stromanurtures the proliferative and invasive neoplastic cells, by providingneovasculature, extracellular matrix components, and stimulatory growthfactors. In this context, CSPG4 plays a major role in tumor-stromaactivation through alterations in cellular adhesion, migration,proliferation, and vascular morphogenesis (Chekenya and Immervoll,2007).

CSPG4 is differentially expressed in human gliomas with higherexpression in high compared to low-grade gliomas (Chekenya et al.,1999). High expression of CSPG4 correlates with multidrug resistancemediated by increased activation of α3β1 integrin/PI3K signaling andtheir downstream targets, promoting cell survival (Chekenya et al.,2008).

CSP-001 was found in the following organs/tissues and cancers:

Brain: —glioblastoma; —secondary glioblastoma (derived from astrocytoma)Colon: —adenocarcinoma (excluding mucinous type), primary;Rectum: —adenocarcinoma, metastasisStomach: —adenocarcinoma (excluding signet ring cell type), primaryKidney: —renal cell carcinoma, cell line; —renal cell carcinoma, clearcell type, metastasis, all secondary sites; —renal cell carcinoma, clearcell type, primary; —renal cell carcinoma, primaryLung: —adenocarcinoma, primary; —adenosquamous carcinoma, primary;—primary cancer; —small cell carcinoma, primary; —squamous cellcarcinoma, primary;Pancreas: —adenocarcinoma, primary; —islet cell tumor, malignant,metastasisProstate: —adenocarcinoma, primarySkin: —metastatic malignant melanoma, lymph node metastasis

Therefore, a pharmaceutical composition containing a peptide accordingto SEQ ID NO:1 is particularly preferred for the treatment of

Brain: —glioblastoma; —secondary glioblastoma (derived from astrocytoma)Colon: —adenocarcinoma (excluding mucinous type), primary;Rectum: —adenocarcinoma, metastasisStomach: —adenocarcinoma (excluding signet ring cell type), primaryKidney: —renal cell carcinoma, cell line; renal cell carcinoma, clearcell type, metastasis, all secondary sites; renal cell carcinoma, clearcell type, primary; renal cell carcinoma, primaryLung: —adenocarcinoma, primary; stage I, —adenosquamous carcinoma,primary; —primary cancer; —small cell carcinoma, primary; —squamous cellcarcinoma, primary;Pancreas: —adenocarcinoma, primary; —islet cell tumor, malignant,metastasisProstate: —adenocarcinoma, primarySkin: —metastatic malignant melanoma, lymph node metastasis

Acyl-CoA Synthetase Bubblegum Family Member 1 (ACSBG1)

The protein encoded by this gene possesses long-chain acyl-CoAsynthetase activity. It is thought to play a central role in brain inactivation of very long-chain fatty acids metabolism and myelinogenesis.Activation of fatty acids by thioesterification to Acetyl-CoA is aprerequisite of most reactions involving this class of molecules.Cancer-specific functions or over-expression has not yet been describedin literature. The expression pattern of ACSBG1 in brain, adrenal gland,testis, and ovary and its function suggests a role of this protein inthe biochemical pathology of X-linked adrenoleukodystrophy (XALD). XALDis a severe, often fatal, neurodegenerative disorder characterized byelevated plasma and tissue levels of saturated very long-chain fattyacids (Asheuer et al., 2005; Pei et al., 2003).

Brevican (BCAN)

Brevican is an extracellular matrix protein that is highly expressed atbirth expressed from birth through 8 years of age and is downregulatedby 20 years of age to low levels that are maintained in the normal adultcortex. A GPI isoform is expressed at uniformly low levels throughoutdevelopment (Gary et al., 2000). Malignant gliomas aggressively invadethe surrounding normal brain which might be mediated by tissue- ortumor-specific extracellular proteins. Thus the extracellular matrix canmodulate, in part, the permissiveness of a tissue to cell movement.Accordingly, the ability of gliomas to modify the ECM of the CNS maymediate the invasiveness of these cells. One ECM molecule that showsdramatic upregulation in gliomas is BCAN, a brain specific chondroitinsulfate proteoglycan. BCAN expression is also upregulated during periodsof increased glial cell motility in development and following braininjury. In glioma an approximately sevenfold increase in expression overnormal levels can be detected (Gary et al., 2000; Gary et al., 1998). Inaddition to upregulation of BCAN in glioma, proteolytic processing ofthe full-length protein also may contribute to invasion (Gary et al.,1998; Nutt et al., 2001). It could be shown that the proteolyticprocessing of BCAN by metalloproteases of the ADAMTS family is anecessary step in mediating its pro-invasive effect in glioma. Themutant, “uncleavable” form of BCAN is unable to enhance glioma cellinvasion in vitro and tumor progression in vivo (Viapiano et al., 2008).mRNA for BCAN is not detectable in normal adult human cortex or in anynonglioma tumor, thus BCAN is considered to be a unique and selectivemarker in glioma (Jaworski et al., 1996). Furthermore, protein analysisdisclosed not only an increased expression of the full-length BCAN butalso the presence of additional, unique isoforms in glioma. Thus,B/b_(Δg) is a full-length product of BCAN mRNA that arises from anincomplete or reduced glycosylation of the core protein. B/b_(Δg) isabsent from the normal adult brain but is found in high-grade gliomasamples (Viapiano et al., 2005).

BCAN has been described as selectively overexpressed in a type ofglioblastoma-derived stem-like tumor cell (Gunther et al., 2008). Thissubtype of stem-like cells showed highest pluripotency andtumorigenicity in vivo.

Chitinase 3-Like 1 (Cartilage Glycoprotein-39) (CHI3L1)

CHI3L1, a member of the “mammalian chitinase-like proteins” is expressedand secreted by several types of solid tumors. It is produced by cancercells and tumor-associated macrophages, exhibits growth factor activityfor cells involved in tissue remodeling processes and might play a rolein cancer cell proliferation, differentiation, survival, invasiveness,metastasis, in angiogenesis and the inflammation and remodeling of theextracellular matrix surrounding the tumor (Johansen et al., 2006;Johansen et al., 2007; Ringsholt et al., 2007). Besides, CHI3L1 is acandidate autoantigen in rheumatoid arthritis. CD4 T cell lines fromhealthy donors directed against CHI3L1 expressed CD25,glucocorticoid-induced tumor necrosis factor receptor, and Foxp3molecules and were capable of suppressing antigen-specific T cellresponses. Responses in 50% of patients with rheumatoid arthritisexhibit polarization toward a proinflammatory T helper 1 phenotype andare significantly less powerful in suppressing antigen-specific recallresponses (van Bilsen et al., 2004).

CHI3L1 is up-regulated by oncostatin M which is known to be induced inthe nervous system as a result of cell stress, is expressed in mosthuman brain tumors and activates the JAK/STAT signaling pathway (Kronaet al., 2007). CHI3L1 expression was also associated with the expressionof p-MAPK, p-mTOR and p-p70S6K in glioblastoma (Pelloski et al., 2006).

In several gene expression studies, CHI3L1 was shown to be more highlyexpressed in glioblastoma compared to normal brain with a range of 3- to62-fold elevation over normal brain (Saidi et al., 2007; Kroes et al.,2007; Shostak et al., 2003; Tanwar et al., 2002). Immunohistochemicalstudies revealed that all cells with a functioning nucleus are capableof expressing CHI3L1 in their cytoplasm but the intensity ofCHI3L1-expression was dependent on cellular activity. Thus cells knownfor exerting a high metabolic activity tended to show the most intensecytoplasmic staining (Ringsholt et al., 2007). Furthermore it could beshown by immunohistochemistry that glioblastomas show strikingly moreCHI3L1 expression than anaplastic oligodendrogliomas (Nutt et al.,2005). Western blot analysis of glioma samples for CHI3L1 protein levelsrevealed substantial elevation in 65% of GBMs and undetectable levels inlower-grade gliomas (grade II and III) or normal brain tissue (Tanwar etal., 2002) In comparison to pilocytic astrocytoma, which does not spreadand can be cured by surgery, only glioblastoma expresses CHI3L1 (Colinet al., 2006).

Serum levels of CHI3L1 are elevated in a variety of malignancies andhave been associated with worse survival. Highest serum levels of CHI3L1were found in patients with metastatic cancer with the shortestrecurrence-free interval and shortest overall survival. Specifically inserum from glioblastoma patients CHI3L1 expression was elevated (Kim etal., 2007; Johansen et al., 2007; Johansen et al., 2006; Junker et al.,2005; Tanwar et al., 2002). GBM patients with active tumor have asignificantly higher level of CHI3L1 than patients with no radiographicevidence of disease. Furthermore there is a significant inverseassociation between CHI3L1 and survival in GBM (Hormigo et al., 2006;Pelloski et al., 2005).

In addition, elevated CHI3L1-expression can be observed in breastcancer, where it correlates with larger tumor size, poorer tumordifferentiation and a worse disease-free survival (Kim et al., 2007;Coskun et al., 2007). Moreover, in squamous cell carcinoma of the headand neck elevated CHI3L1 serum levels were detected in 53%. Patientswith high serum CHI3L1 have shorter survival than patients with normalserum CHI3L1 (33 vs. 84 months) (Roslind et al., 2008).

Patients suffering from prostate cancer showed significantly higherserum levels of CHI3L1 in comparison to patients with BPH or healthypersons (Kucur et al., 2008).

CAP-GLY Domain Containing Linker Protein 2 (CLIP2)

The protein encoded by CLIP2 belongs to the family of cytoplasmic linkerproteins, which have been proposed to mediate the interaction betweenspecific membranous organelles and microtubules. CLIP2 was found toassociate with both microtubules and an organelle called the dendriticlamellar body (general information from the NCBI-web page).

CLIP2 localizes to the ends of tyrosinated microtubules but not to theends of detyrosinated microtubules. Tubulin-tyrosine ligase (TTL), theenzyme that catalyzes the addition of a C-terminal tyrosine residue toalpha-tubulin in the tubulin tyrosination cycle, is involved in tumorprogression and has a vital role in neuronal organization (Penis et al.,2006). One study of genomic DNA from frozen sections of 30 cases ofprimary glioblastomas by GenoSensor Array 300 characterized geneamplifications, gene deletions, and chromosomal information in the wholegenome. Genes that were frequently amplified included=CLIP2 (63.3%),EGFR (53.3%), IL6 (53.3%), ABCB1 (MDR1) (36.7%), and PDGFRA (26.7%)(Suzuki et al., 2004).

Solute Carrier Organic Anion Transporter Family, Member 1C1 (SLCO1C1)

SLCO1C1 is selectively expressed at the blood-brain barrier (Chu et al.,2008). SLCO1C1 has selective substrate preference and may play animportant role in the disposition of thyroid hormones in brain andtestis (Pizzagalli et al., 2002). SLCO1C1 was not specificallydetectable by immunofluorescence. SLCO1A2 and SLCO2B1 were detectable byimmunofluorescence microscopy in the luminal membrane of endothelialcells forming the blood-brain barrier and the blood-tumor barrier, butnot in the glioma cells (Bronger et al., 2005).

Dystrobrevin, Alpha (DTNA)

Alpha-dystrobrevin has been described primarily as a cytoplasmiccomponent of the dystrophin-glycoprotein complex in skeletal musclecells. Isoforms of DTNA show different localization in cells andtissues; at basolateral membranes in epithelial cells, dystrobrevinsmediate contact with the extracellular matrix, peripheral andtransmembrane proteins and the filamentous actin cytoskeleton. Besidetheir structural role, DTNAs are assumed to be important in cellsignalling and cell differentiation and are associated with the tightjunctions during their reorganization (Sjo et al., 2005). DTNA may beinvolved in the formation and stability of synapses as well as theclustering of nicotinic acetylcholine receptors.

Epidermal Growth Factor Receptor (Erythroblastic Leukemia Viral(v-Erb-b) Oncogene Homolog, Avian) (EGFR)

A recent area of interest is the epidermal growth factor receptor(EGFR), since its abnormalities are one of the most common molecularaberrations in glioblastoma. Especially EGFRvIII (epidermal growthfactor receptor variant III) is an oncogenic, constitutively activemutant form of the EGFR that is commonly expressed in glioblastoma(Zawrocki and Biernat, 2005). EGFR is involved in the activation of anumber of pathways that regulate the phenotype of progenitor cells.Activated EGFR tyrosine kinase activity enhances neural stem cellmigration, proliferation and survival. As EGFR signaling is also knownto play a role in glioblastoma, it can be concluded that glioblastomaderives from a cancer stem cell and that EGFR signals are commonlyaltered in these precursor cells (Yuso-Sacido et al., 2006).

Primary glioblastomas arise de novo in older patients and oftenoverexpress EGFR. EGFR overexpression correlates with increasedangiogenesis, edema and invasion (Aghi et al., 2005). Furthermore,EGFR-amplified glioblastomas are radiation resistant (Barker et al.,2001) and recur more rapidly after treatment (Schlegel et al., 1994).

GBM is the only nonepithelial human tumor for which excessive activationof EGFR has been linked to tumor growth and patient survival, and EGFRactivation promotes GBM infiltration in vitro (Penar et al., 1997).

EGFR is the proto-oncogene of erbB. Overexpression of EGFR can augmentcell growth because of increased formation of active ligand:receptorcomplexes. Gene amplification is the mechanism underlying overexpressionof EGF receptors in GBM tumors (Thompson and Gill, 1985). The EGFR geneon chromosome 7 is known to gain in copy number frequently in high-gradegliomas (Okada et al., 2007). Depletion of EGFR by short interferenceRNA abolishes the tumorigenesis of glioblastoma cells (Huang et al.,2007).

EGFR overexpression is detected in 40-70% of GBM whereas pilocytic,low-grade or anaplastic astrocytoma are invariably EGFR negative.(Agosti et al., 1992; Schwechheimer et al., 1995; Eppenberger andMueller, 1994; Huncharek and Kupelnick, 2000; Liu et al., 2006a). Highserum levels of EGFR indicate reduced survival (Quaranta et al., 2007).Furthermore, it was shown that long-term survivors with high gradeastrocytomas are EGFRvIII negative (Liang et al., 2008).

Notch-1 up-regulates EGFR expression and correlations between levels ofEGFR and Notch-1 mRNA can be found in primary high-grade human gliomas(Purow et al., 2008). EGFR itself is involved in constitutive activationof c-Jun NH2-terminal kinase (JNK), which contributes to proliferation,survival and tumorigenesis in some tumors, including gliomas (Li et al.,2008a).

Although EGFRvIII is only expressed by a small percentage of gliomacells, most of the cells exhibit a transformed phenotype. It was shownthat EGFRvIII expression in indolent glioma cells stimulates formationof lipid-raft related microvesicles containing EGFRvIII which arereleased to cellular surroundings and can merge with the plasmamembranes of cancer cells lacking EGFRvIII leading to the transfer ofoncogenic activity (Al-Nedawi et al., 2008).

Fatty Acid Binding Protein 7, Brain (FABP7)

Fatty acid-binding proteins (FABPs) are cytosolic 14-15 kDa proteins,which are supposed to be involved in fatty acid (FA) uptake, transport,and targeting. They may modulate FA concentration and in this wayinfluence function of enzymes, membranes, ion channels and receptors,and gene expression and cellular growth and differentiation. Nine FABPtypes can be discerned with a specific tissue distribution. In spite of30-70% amino acid sequence identity, they have a similar tertiary,beta-clam structure in which the FA is bound. Nervous tissue containsfour FABP types with a distinct spatio-temporal distribution (Veerkampand Zimmerman, 2001). FABP7 is highly expressed in the developing brainand retina and its expression decreases significantly in the adult CNS(Godbout et al., 1998). Based on in vitro results, it has been suggestedthat FABP7 is required for the establishment of the radial glial systemof the developing brain (Mita et al., 2007). In normal brain FABP7protein is barely detectable but shows moderate to strong nuclear andcytoplasmic expression in several GBMs. FABP7-transfected cells display5-fold greater migration than control cells. Thus, the shorter overallsurvival associated with FABP7 overexpression especially in glioblastomamay be due to increased migration and invasion of tumor cells into thesurrounding brain parenchyma (Liang et al., 2005). Nuclear translocationof FABP7 is specifically related to EGFR amplification and more invasivetumors (Kaloshi et al., 2007). Thus, nuclear FABP7 may be induced byEGFR activation to promote migration of GBM tumor cells (Liang et al.,2006).

FABP7 expression has also been shown to be a marker for renal cellcarcinoma. FABP7-expression can be detected only in carcinoma tissuesbut not in noncancerous parts of kidney samples (Teratani et al., 2007).The expression of FABP7 in renal cell carcinoma was shown to be 20-foldhigher in the tumor in comparison to normal kidney tissue (Domoto etal., 2007; Buchner et al., 2007). It was also shown that FABP7 isfrequently expressed in melanoma where it may be involved in cellproliferation and invasion (Goto et al., 2006).

Glial Fibrillary Acidic Protein (GFAP)

GFAP encodes one of the major intermediate filament proteins of matureastrocytes. It is used as a marker to distinguish astrocytes from otherglial cells during development. Mutations in this gene cause Alexanderdisease, a rare disorder of astrocytes in the central nervous system. Anadditional transcript variant has been described, but its full lengthsequence has not been determined. Increased levels have been reported inautistic brains whereas brains from people with severe depression showeddecreased GFAP.

Brains from primates that developed de novo tumors ten years after wholebrain radiation were analyzed. Tumor precursors demonstrated cellularatypia and mitoses, and were negative for tumor-associated markers GFAP,EGFR and p53 (Lubensky et al., 2006).

In astrocytic neoplasms the number of GFAP positive cells and theintensity of the stain were directly proportional to the degree ofmalignancy. All the 3 cases of oligodendroglioma showed a negativereaction to GFAP (Reyaz et al., 2005). Pure oligodendrogliomas areimmunohistologically negative for GFAP (Mokhtari et al., 2005). GFAPserum levels in patients with high grade glioma demonstrated a linearcorrelation to tumour volume (Brommeland et al., 2007). Even among GBpatients a significant correlation between tumour volume, tumournecrosis volume, the amount of necrotic GFAP positive cells and serumGFAP level can be detected (Jung et al., 2007).

Following treatment of glioblastoma cell lines with the histonedeacetylase inhibitor 4-phenylbutyrate, increased concentrations ofnon-phosphorylated GFAP were seen, while phosphorylated isoformsremained intact (Asklund et al., 2004).

In a glioblastoma cell line treated with TGF-alpha, GFAP genetranscription, mRNA level, and specific protein synthesis decreased byapproximately 50% (Zhou and Skalli, 2000).

Technically, the GFAP promoter is frequently used as a tool in mousemodels to induce the expression of desired proteins specifically in thenervous system.

Pancreatic islets of Langerhans are enveloped by peri-islet Schwanncells (pSC), which express GFAP. Autoimmune targeting of pancreaticnervous system tissue elements seems to be an integral, early part ofnatural type 1 diabetes (Winer et al., 2003). This pancreatic expressionis not reflected by immatics or external gene expression data from bulktissues. GFAP-001 has been published as an epitope against which type 1diabetic patients as well as their non-diabetic relatives with antibodyresponses against diabetes autoantigens (increased risk for diabetes)showed enhanced reactivity of granzyme B-secreting CTLs (ex vivoELISPOT) compared with healthy donors (Standifer et al., 2006).

Interestingly, an inverse correlation between the manifestation ofautoimmune diseases, especially diabetes, and the risk of gliomadevelopment seems to exist (Aronson and Aronson, 1965; Schlehofer etal., 1999; Brenner et al., 2002; Schwartzbaum et al., 2003; Schwartzbaumet al., 2005).

G Protein-Coupled Receptor 56 (GPR56)

GPR56 is an atypical G protein-coupled receptor (GPCR) with an unusuallylarge N-terminal extracellular region, which contains a longSer/Thr-rich region forming a mucin-like stalk and due to this feature,GPR56 is thought to play a role in cell-cell, or cell-matrixinteractions. Together with the high level of mRNA expression and itswide distribution, a possible role for this receptor in cell-cellinteraction processes has been suggested (Liu et al., 1999). GPR56belongs to the GPCR of the secretin family which has a role in thedevelopment of neural progenitor cells and has been linked todevelopmental malformations of the human brain. Higher GPR56 expressionis correlated with the cellular transformation phenotypes of severalcancer tissues compared with their normal counterparts, implying apotential oncogenic function. RNA interference-mediated GPR56 silencingresults in apoptosis induction and reduced anchorage-independent growthof cancer cells via increased anoikis. GPR56 silencing also reduces celladhesion to the extracellular matrix (Ke et al., 2007). Upregulation ofGPR56 has been demonstrated in glioblastoma multiforme using functionalgenomics. Immunohistochemistry studies confirmed the expression of GPR56in a majority of glioblastoma/astrocytoma tumor samples withundetectable levels of expression in normal adult brain (Shashidhar etal., 2005). In pancreatic cancer cells, GPR56 mRNA is expressed at highlevels whereas GPR56 protein is either negligible or undetectable inthese cells suggesting that the expression of GPR56 protein issuppressed in human pancreatic cancer cells (Huang et al., 2008).Earlier studies concerning metastasis showed that GPR56 is markedlydown-regulated in highly metastatic variants from a human melanoma cellline implying that overexpression of GPR56 suppresses tumor growth andmetastasis. This growth suppression is thought to be mediated byinteraction of GPR56 with tissue transglutaminase (TG2), a widespreadcomponent of tissue and stroma, which has been implicated in suppressionof tumor progression itself (Xu et al., 2006; Xu and Hynes, 2007).Another inhibitory impact of GPR56 has been reported for the migrationof neural progenitor cells (NPCs). GPR56 is highly expressed in NPCs andprobably participates in the regulation of NPC movement through theGalpha(12/13) and Rho signaling pathway, suggesting that GPR56 plays animportant role in the development of the central nervous system (Iguchiet al., 2008).

Glutamate Receptor, Ionotrophic, AMPA 4 (GRIA4)

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate(AMPA)-type glutamatereceptors (AMPARs) mediate fast neurotransmission in excitatory synapsesin the CNS and are composed of subunits taken from a set of fourproteins, GluR1 through GluR4 (GRIA4).

GRIA4 subunits are ubiquitously expressed in human glioblastoma cells,operating as Ca²⁺-permeable AMPARs. Conversion to Ca²⁺-impermeablereceptors inhibits cell locomotion and induces apoptosis whereasoverexpression of Ca²⁺-permeable AMPA receptors facilitates migrationand proliferation of the tumor cells. Therefore Ca²⁺-permeable AMPAreceptors seem to have crucial roles in growth of glioblastoma (Ishiuchiet al., 2002).

Insulin-Like Growth Factor 2 mRNA Binding Protein 3 (IGF2BP3)

IGF2BP3 is a member of the insulin-like growth factor-II mRNA-bindingprotein family, implicated in mRNA localization, turnover andtranslational control. The encoded protein contains several KH domains,which are important in RNA binding and are known to be involved in RNAsynthesis and metabolism. It is expressed mainly during embryonicdevelopment and in some tumors. Thus, IGF2BP3 is considered to be anoncofetal protein (Liao et al., 2005). Specific information aboutIGF2BP3 expression in glioblastoma was not found, but IGF2BP3 isdescribed to be overexpressed in several other malignancies. Thus,IGF2BP3 is expressed in 30% of 716 analyzed clear cell renal cellcarcinoma specimen. In this study, its expression was associated withadvanced stage and grade of primary tumors as well as other adversefeatures including coagulative tumor necrosis and sarcomatoiddifferentiation. Furthermore, positive IGF2BP3 expression was associatedwith a 5-10 fold increased risk of distant metastases and with a 42%-50%increase in the risk of death from RCC, suggesting that IGF2BP3expression represents an independent predictor of aggressive clear cellrenal cell carcinoma tumor behavior (Hoffmann et al., 2008; Jiang etal., 2006; Jiang et al., 2008). IGF2BP3 expression was also detectablein malignant melanoma in comparison to benign nevi, where no expressionwas to be determined, even when dysplastic features are present (Pryoret al., 2008). In endometrial cancer, expression of IGF2BP3 is closelyassociated with type II endometrial cancer and an aggressive histologicphenotype among endometrial neoplastic lesions (Zheng et al., 2008). In20 patients suffering from esophageal squamous cell carcinoma, inductionof specific T-cell responses in TILs, regional lymph node lymphocytesand peripheral blood lymphocytes against a HLA-A2402-restricted epitopepeptide from IGF2BP3 could be observed in 40% of all cases (Mizukami etal., 2008). IGF2BP3 is also highly expressed in pancreatic carcinomas.In 2 studies >90% of pancreatic tumor tissue samples showed IGF2BP3expression after immunostaining whereas non-neoplastic pancreatictissues were negative for IGF2BP3. Furthermore, IGF2BP3 mRNA wasoverexpressed in pancreatic carcinomas in comparison to non-neoplastictissue samples and the expression increased progressively with tumorstage (Yantiss et al., 2005; Yantiss et al., 2008). IGF2BP3 expressionwas also found to be significantly increased in high-grade urothelialtumors while it is generally not expressed in benign urothelium orlow-grade urothelial tumors. Moreover, patients with IGF2BP3-positivetumors have a much lower progression-free survival and disease-freesurvival rate than those with IGF2BP3-negative tumors. IGF2BP3-positivepatients with superficial invasive urothelial carcinoma at initialdiagnosis also went on to develop metastases, whereas no metastasis wasfound in IGF2BP3-negative patients. In addition, data from these studiessuggested that IGF2BP3 may be involved in the progression of urothelialtumors from low grade to high grade in both papillary and flat lesions(Li et al., 2008b; Sitnikova et al., 2008).

Megalencephalic Leukoencephalopathy with Subcortical Cysts 1 (MLC1)

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is anautosomal recessive cerebral white matter disorder in children. MLC iscaused by mutations in the gene MLC1 (Ilja Boor et al., 2006). Accordingto the understanding of the inventors, no reports about any associationof MLC1 with brain tumors are found in the literature.

One paper investigated the cellular and regional distribution of MLC1 inmouse brain (Schmitt et al., 2003). Highest MLC1 expression was foundduring the pre- and perinatal period in multipotential neural precursorcells. In the adult mouse brain MLC1 mRNA was exclusively detected inglial cells. In contrast to developing and mature astrocytes,oligodendrocytes were devoid of MLC1 expression.

Nestin (NES)

During development, there is extensive expression of the intermediatefilament nestin in neuroepithelial cells in the ventricular layer at 11weeks post-conceptional age in all parts of the CNS, whereas nestinimmunoreactivity diminishes during the second and third trimesters(Takano and Becker, 1997; Lendahl et al., 1990; Zimmerman et al., 1994;Tohyama et al., 1992). During or after migration away from theproliferative ventricular layer, nestin expression is sharplydownregulated in post-mitotic neurons (Arnold and Trojanowski, 1996).Nestin-staining of non-neoplastic adult human brain tissue showed onlyweak staining of a very small number of endothelial cells (Dahlstrand etal., 1992). Nestin can be re-expressed during neoplastic transformation(Veselska et al., 2006). In glioma tissues, nestin immunoreactivityoccurs only in tumor cells and the quantity of nestin produced increasesas the grade of glioma becomes more malignant toward glioblastoma.Glioblastomas (malignancy grade IV) express the highest incidence ofnestin-positive cells and in general the highest levels of nestinstaining Nestin expression can be detected in tumor cells of varioustypes of primary CNS tumors, which are of neuroectodermal origin, butnot in metastasizing carcinoma cells (Dahlstrand et al., 1992; Tohyamaet al., 1992). Nestin is almost not expressed in diffuse astrocytomas,variably expressed in anaplastic astrocytomas and strongly andirregularly expressed in glioblastomas, where its distribution varies ina complementary way with GFAP and Vimentin (Schiffer et al., 2006).Clinically, nestin-negative CNS germ cell tumors did not exhibitdissemination, while all tumors that exhibited dissemination alsostrongly expressed nestin protein (Sakurada et al., 2007).

Tumor cells strongly expressing nestin are often located close to bloodvessels (Dahlstrand et al., 1992), (Kurihara et al., 2000; Sugawara etal., 2002; Florenes et al., 1994; Strojnik et al., 2007) and nestinexpression by activated endothelium has been suggested as anangiogenesis marker (Teranishi et al., 2007; Maderna et al., 2007; Amohet al., 2005; Mokry et al., 2004).

GBM comprises transformed precursors that bear the full complement offunctional characteristics expected from stem cells, including thecapacity for tumor generation. These cells can establish GBM even uponserial transplantation and can therefore be identified as tumor neuralstem cells (Galli et al., 2004). These cells belong to the CD133+ cellsubpopulation from human brain tumors and co-express the NSC markernestin, but not differentiated neural lineage markers (Singh et al.,2004b; Singh et al., 2003; Singh et al., 2004a; Mao et al., 2007). Thepresence of a CD133+/nestin+ population in brain tumors suggests that anormal neural stem cell may be the cell of origin for gliomas (Shiras etal., 2007). As Notch signaling is active in brain tumor and stem cells,it has been shown that the nestin promoter is activated in culturethrough Notch activity (Shih and Holland, 2006).

Transfecting the rat astrocytoma C6 cell line with nestin siRNA duplexrevealed an effective suppression influence of nestin siRNA on cellgrowth of cultured astrocytoma cells in a dose-dependent manner (Wei etal., 2008).

Nestin expression has also been reported for cancer stem cells inprostate (Gu et al., 2007; Gipp et al., 2007) and pancreatic cancer(Carriere et al., 2007) as well as melanoma (Klein et al., 2007).Furthermore, nestin is also expressed in the following tumors: GIST(Tsujimura et al., 2001; Sarlomo-Rikala et al., 2002), melanomas(Florenes et al., 1994; Brychtova et al., 2007), Colorectal cancer(Teranishi et al., 2007) and pancreatic tumors (Ohike et al., 2007;Kleeberger et al., 2007).

Nestin expression can also be found in various normal tissues: Nestinexpression has been reported in podocytes of normal mature human kidneyglomeruli. In normal conditions nestin is expressed in severalglomerular cell types at early stages of development and becomesconfined to podocytes in mature glomeruli (Ishizaki et al., 2006),indicating that nestin is critical for some aspect of podocyte function.Adult glomeruli display nestin immunoreactivity in vimentin-expressingcells with the podocyte morphology (Bertelli et al., 2007). Possiblynestin serves through an interaction with vimentin to bolster themechanical strength of podocytes which experience high tensile stressduring glomerular filtration (Perry et al., 2007). Thus, in humankidney, nestin is expressed from the first steps of glomerulogenesiswithin podocytes, mesangial, and endothelial cells. This expression isthen restricted to podocytes in mature glomeruli and can not be detectedin other structures of the adult human kidney (Su et al., 2007).Immunohistochemistry revealed constant nestin expression in the cortexof normal human adrenal glands. Nestin expressing cells are prevalentlylocated in the zona reticularis whereas adrenal carcinomas display avariable number of nestin-immunoreactive cells (Toti et al., 2005).

Nestin expression is also reported from interstitial cells of Cajal(ICC) in the normal gastrointestinal tract. Thus most intramuscular ICCin antrum and all myenteric ICC in small intestine arenestin-immunoreactive as well as some myenteric ICC and most ICC in thecircular musculature of the colon (Vanderwinden et al., 2002). Inpancreas, nestin-immunoreactive cells can be found in islets and in theexocrine portion. In the area of big pancreatic ducts, nestin-positivecells represent small capillaries scattered in the connective tissuesurrounding the duct epithelium. Thus, nestin is expressed by vascularendothelial cells in human pancreas (Klein et al., 2003). In the isletsthemselves islet progenitor cells that express nestin can be found. Itis hypothesized that these nestin-positive islet-derived progenitorcells are a distinct population of cells that reside within pancreaticislets and may participate in the neogenesis of islet endocrine cells(Zulewski et al., 2001). In the adult normal liver a population of humanliver stem cells that are positive for vimentin and nestin can beisolated (Herrera et al., 2006). In cell culture assays, analysis ofcytoskeleton and matrix composition by immunostaining revealed thatfetal lung- and adult marrow-derived cells express vimentin and nestinproteins in intermediate filaments (Sabatini et al., 2005). In youngpermanent teeth, nestin is found in functional odontoblasts. Itsexpression is down-regulated and nestin is absent from older permanentteeth while it is up-regulated again in carious and injured teeth (Aboutet al., 2000).

Nestin-expressing adult stem cells can also be found in the perilumenalregion of the mature anterior pituitary and, using genetic induciblefate mapping, it was demonstrated that they serve to generate subsets ofall six terminally differentiated endocrine cell types of the pituitarygland. These stem cells, while not playing a significant role inorganogenesis, undergo postnatal expansion and start producingdifferentiated progeny, which colonize the organ that initially entirelyconsisted of differentiated cells derived from embryonic precursors(Gleiberman et al., 2008).

Nuclear Receptor Subfamily 2, Group E, Member 1 (NR2E1)

NR2E1 (TLX) is a transcription factor that is essential for neural stemcell proliferation and self-renewal by recruiting histone deacetylases(HDACs) to its downstream target genes to repress their transcription,which in turn regulates neural stem cell proliferation. Recruitment ofHDACs leads to transcriptional repression of TLX target genes, thecyclin-dependent kinase inhibitor, p21(CIP1/WAF1)(p21), and the tumorsuppressor gene, PTEN (Sun et al., 2007). The TLX/HOX11 subfamily ofdivergent homeobox genes are involved in various aspects ofembryogenesis and, in the case of TLX1/HOX11 and TLX3/HOX11L2, featureprominently as oncogenes in human T-cell acute lymphoblastic leukemia(Dixon et al., 2007). NR2E1 underlies a fundamental developmentalprogram of retinal organization and controls the generation of theproper numbers of retinal progenies and development of glial cellsduring the protracted period of retinogenesis (Miyawaki et al., 2004).No glioblastoma specific information found.

Neuronal Cell Adhesion Molecule (NRCAM)

Human NRCAM (Neuroglia related Cell Adhesion Molecule) is over expressedin glioblastoma multiforme tissue (GMT) as compared to normal braintissue (NBT). NRCAM is described as single-pass type I transmembraneprotein interacting with ankyrin. Antisense hNRCAM caused reduction inthe native hNRCAM expression, changes in cell morphology, reduced cellproliferation rate and lengthening of the cell cycle. Furthermore,antisense hNRCAM overexpression in these cells caused extensivereduction in the number of soft agar colonies and invasion through extracellular matrix (ECM) gel in vitro. Subcutaneous injection of antisensehNRCAM overexpressing glioblastoma cells into nude mice caused completeinhibition of tumor formation as compared to vector only transfectedcells. Intra-tumoral inoculation of antisense hNRCAM expressing plasmidalso caused slow tumor growth in nude mice in vivo (Sehgal et al.,1999). Gene-specific RT-PCR analysis indicated that hNRCAM isover-expressed in high-grade astrocytomas, gliomas and glioblastomatumor tissues as compared to normal brain tissue (Sehgal et al., 1998).NRCAM, a cell-cell adhesion molecule of the immunoglobulin-like celladhesion molecule family, known for its function in neuronal outgrowthand guidance, was recently identified as a target gene of beta-cateninsignaling in human melanoma and colon carcinoma cells and tissue.Retrovirally mediated transduction of NRCAM into fibroblasts inducescell motility and tumorigenesis (Conacci-Sorrell et al., 2005).Induction of NRCAM transcription by beta-catenin or plakoglobin plays arole in melanoma and colon cancer tumorigenesis, probably by promotingcell growth and motility (Conacci-Sorrell et al., 2002). Also othertargets in beta-catenin signalling are upregulated—such as MYC (Liu etal., 2008). NrCAM is overexpressed in human papillary thyroid carcinomasat the mRNA and protein levels, whatever the tumor stage (Gorka et al.,2007).

Overexpression of NRCAM mRNA in tumors is associated with highproliferation indices and was associated with a poor outcome inependymomas (Zangen et al., 2007).

Podoplanin (PDPN)

PDPN is a mucin-like type-I integral membrane glycoprotein with diversedistribution in human tissues. It is involved in cancer cell migration,invasion, metastasis and malignant progression and is involved inplatelet aggregation. CLEC-2 is the first identified pathophysiologicalreceptor of podoplanin (Kato et al., 2008). 115 glioblastomas wereinvestigated using immunohistochemistry with an anti-PDPN antibody. 47%of glioblastomas expressed PDPN on surface cells, especially aroundnecrotic areas and proliferating endothelial cells. Furthermore, PDPNmRNA and protein expression were markedly higher in glioblastoma than inanaplastic astrocytomas suggesting that PDPN expression might beassociated with malignancy of astrocytic (Mishima et al., 2006). PDPNwas also shown to be expressed in 82.9% of glioblastomas (29/35) inanother analyses (Shibahara et al., 2006). In a study investigating PDPNexpression and platelet-aggregating activites of glioblastoma celllines, LN319 highly expressed PDPN and induced platelet aggregation.NZ-1, a highly reactive anti-PDPN antibody, neutralized plateletaggregation by LN319 suggesting that PDPN is a main reason for plateletaggregation induced by (Kato et al., 2006). PDPN gene expression levelswere significantly higher in glioblastomas than in non-neoplastic whitematter, which was confirmed by immunohistochemistry (Scrideli et al.,2008). PDPN is specifically expressed by lymphatic but not bloodvascular endothelial cells in culture and in tumor-associatedlymphangiogenesis. Although PDPN was primarily absent from normal humanepidermis, its expression was strongly induced in 22 of 28 squamous cellcarcinomas suggesting a role for PDPN in tumor progression (Schacht etal., 2005). PDPN is upregulated in the invasive front of a number ofhuman carcinomas. Investigation of PDPN expression in cultured humanbreast cancer cells, in a mouse model of pancreatic beta cellcarcinogenesis, and in human cancer biopsies indicated that PDPNpromotes tumor cell invasion in vitro and in vivo. PDPN inducescollective cell migration by filopodia formation via the downregulationof the activities of small Rho family GTPases. In conclusion, PDPNinduces an alternative pathway of tumor cell invasion in the absence ofepithelial-mesenchymal transition (Wicki et al., 2006) PDPN expressionlevel was enhanced in most colorectal tumor patients (Kato et al., 2003)TGF-beta is supposed to be a physiological regulator of PDPN in tumorcells (Suzuki et al., 2008) PDPN is expressed by cancer cells derivedfrom esophagus, lung, liver, colon and breast as well as lymphaticendothelial cells (Kono et al., 2007).

Tenascin C (Hexabrachion) (TNC)

The expression of the extracellular matrix glycoprotein TNC inglioblastoma but not in normal brain and its association withglioblastoma-proliferative endothelium basement membranes suggestedalready in 1983 that TNC may be a useful marker of gliomas (Bourdon etal., 1983). During tumor progression, the ECM of tumor tissues isremodeled and now provides an environment that is more conductive fortumor progression, of which angiogenesis is a crucial step (Carnemollaet al., 1999). TNC is overexpressed in tumor vessels that have a highproliferative index which indicates that TNC is involved in neoplasticangiogenesis (Kim et al., 2000). In tumors, TNC-expression can beinduced by hypoxia (Lal et al., 2001). TNC induction is mediated byTGF-beta1, providing a mechanism for the invasion of high-grade gliomasinto healthy parenchyma (Hau et al., 2006). Also, TNC overexpression isa consequence of the specific activation of the tenascin-C gene promoterby gastrin, which is known to significantly modulate the migration ofhuman glioblastoma cells (Kucharczak et al., 2001). TNC down-regulatestropomyosin-1 and thus destabilizes actin stress fibers. It additionallycauses down-regulation of the Wnt inhibitor Dickkopf1. As reducedtropomyosin-1 expression and increased Wnt signaling are closely linkedto transformation and tumorigenesis, TNC specifically modulates thesesignaling pathways to enhance proliferation of glioma cells (Ruiz etal., 2004).

Perivascular staining of TNC around tumor-supplying blood vessels isobserved in glioblastoma tissues, whereas in WHOII and III gliomas,perivascular TNC staining is less frequent, indicating that theintensity of TNC staining correlates with the tumor grade and thestrongest staining indicates poor prognosis (Herold-Mende et al., 2002;Zukiel et al., 2006). Highest TNC-expression is observed in connectivetissue surrounding tumors (Chiquet-Ehrismann and Tucker, 2004). TNC alsocontributes to the generation of a stem cell niche within thesubventricular zone (SVZ), acting to orchestrate growth factor signalingto accelerate neural stem cell development. TNC is essential for thetimely expression of the EGFR in neural stem cells and enhances FGF2signalling. The predominant effect of TNC on cells in the SVZ is theregulation of developmental progression (Garcion et al., 2004). TNC isthe strongest inducer of directed human NSC migration (haptotaxis). Thetumor-produced ECM thus provides a permissive environment for NSCtropism to disseminated tumor cells (Ziu et al., 2006).

The TNC pathway also plays an important role in mammary tumor growth andmetastases. Thus, signaling blockade or knockdown of TNC in MDA-MB-435cells resulted in a significant impairment of cell migration andanchorage-independent cell proliferation. Mice injected with clonalMDA-MB-435 cells with reduced expression of TNC demonstrated asignificant decrease in primary tumor growth as well as a decrease intumor relapse after surgical removal of the primary tumor and a decreasein the incidence of lung metastasis (Calvo et al., 2008).

Survivin (BIRC5)

Expression of BIRC5 (survivin), a member of the inhibitor of apoptosisprotein (IAP) family, is elevated in fetal tissues and in various humancancers, with greatly reduced expression in adult normal differentiatedtissues, particularly if their proliferation index is low. Survivinseems to be capable of regulating both cellular proliferation andapoptotic cell death. Although survivin is usually located in the cellcytoplasmic region and associated with poor prognosis in cancer, nuclearlocalization, indicative of favorable prognosis, has also been reported(O'Driscoll et al., 2003). Regulation of and through survivin has beendescribed by several mechanisms. Survivin seems to be associated withthe molecular chaperone Hsp60. In vivo, Hsp60 is abundantly expressed inprimary human tumors as compared with matched normal tissues. Acuteablation of Hsp60 by small interfering RNA destabilizes themitochondrial pool of survivin, induces mitochondrial dysfunction, andactivates caspase-dependent apoptosis (Ghosh et al., 2008). Furthermore,Ras inhibition results in release of the survivin “brake” on apoptosisand in activation of the mitochondrial apoptotic pathway. Especially inglioblastoma, resistance to apoptosis can be abolished by a Rasinhibitor that targets survivin (Blum et al., 2006). There also seems tobe a correlation between NF-kappaB hyperactivity in gliomas andhyperexpression of survivin, one of NF-kappaB target genes. Thus,NF-kappaB-activated anti-apoptotic genes are hyperexpressed in tumorsamples. Especially in glioblastoma, very high levels of survivinexpression are detectable (Angileri et al., 2008). It is suggested thatsurvivin overexpression in brain gliomas might play an important role inmalignant proliferation, anti-apoptosis and angiogenesis (Zhen et al.,2005; Liu et al., 2006b). Several analyses were performed to studysurvivin expression and its impact on survival in glioblastoma. Tosummarize, survivin expression, especially the simultaneous expressionin nucleus and cytoplasm in astrocytic tumors was significantlyassociated with malignancy grade (with highest survivin expression inglioblastoma) and shorter overall survival times compared with patientswho had survivin-negative tumors (Kajiwara et al., 2003; Saito et al.,2007; Uematsu et al., 2005; Mellai et al., 2008; Grunda et al., 2006;Xie et al., 2006; Sasaki et al., 2002; Chakravarti et al., 2002).

Survivin-overexpression has also been described for other tumorentities. In breast cancer, survivin expression is associated withhigher grade and shorter disease-free survival (Yamashita et al., 2007;Al-Joudi et al., 2007; Span et al., 2004). In esophageal cancer celllines, the promoter activity of survivin was shown to be 28.5 foldhigher than in normal tissues (Sato et al., 2006). In colorectal cancer,survivin expression is also associated with pathological grade and lymphnode metastasis (Tan et al., 2005). The aggressiveness of clear cellrenal cell carcinoma was shown to be associated with survivinexpression. Furthermore, expression of survivin is inversely associatedwith cancer-specific survival (Kosari et al., 2005). Survivin expressioncan be detected in a panel of keratinocytic neoplasms andhyperproliferative skin lesions but not in normal skin (Bowen et al.,2004). In pancreatic cancer cell lines, survivin was amplified in 58% ofthe tested cell lines (Mahlamaki et al., 2002). In squamous cellcarcinoma, survivin expression can help to identify cases with moreaggressive and invasive clinical phenotype (Lo et al., 2001).

As survivin is such a promising target for cancer therapy, studies usingsurvivin-derived peptides showed that survivin is immunogenic in tumorpatients by eliciting CD8+ T cell-mediated responses. In addition,survivin specifically stimulated CD4+ T-cell reactivity in peripheralblood lymphocytes from the same patients (Casati et al., 2003; Piescheet al., 2007).

Survivin (SVN, BIRO) is overexpressed in a multitude of cancer entities.Thus, in general, overexpression of survivin is thought to be associatedwith shorter overall-survival and higher malignancy grades.

The present invention further relates to particular marker proteins thatcan be used in the prognosis of glioblastoma. Further, the presentinvention further relates to the use of these novel targets for cancertreatment.

As provided herein, the proteins GFAP, FABP7, DTNA, NR2E1, SLCO1C1,CHI3L1, ACSBG1, IGF2BP3, NLGN4X, MLC1, NRCAM, BCAN, EGFR, PDPN, NES, andCLIP2 are highly over-expressed in glioblastomas compared with normalbrain and other vital tissues (e.g. liver kidney, heart). The proteinsGRP56, CSPG4, NRCAM, TNC, BIRC5, CLIP2, NES, PDPN, EGFR, BCAN, and GRIA4are shown to have an important role in tumorigenesis as they are ininvolved in malignant transformation, cell growth, proliferation,angiogenesis or invasion into normal tissue.

The proteins NES, TNC, BIRC5, EGFR are associated with cancer stem cellsor stem cell niches in glioblastoma. Cancer stem cells are a tumor cellsubpopulation with self-renewing potential required for sustained tumorgrowth. These cells reside in specialized and highly organizedstructures, so called cancer stem cell niches that are required for themaintenance of the self-renewing potential of cancer stem cells.Overexpression of the proteins BIRC5, NRCAM, IGF2BP3 in tumors has beenshown to be associated with advanced disease stages and poor prognosisfor the patients.

BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and PDPN, are shown to play animportant role in tissue remodeling required for tumor growth in thenervous system. Therefore, the expression of BCA, CLIP2, DTNA, NLGNAX,NR2E1, NRCAM and PDPN can be used as a marker to distinguishglioblastoma from other forms of cancer.

Thus, the present invention provides methods of identifying an animal,preferably a human that is likely to have glioblastoma. In oneembodiment the likelihood determined is from 80% to 100%. One suchmethod comprises determining the level of at least one of the proteinsBCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and PDPN in a tumor sample fromthe animal subject. In one embodiment, the sample is obtained by radicalsurgery. In another embodiment, the sample is obtained by needle biopsy.

When the level of BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM or PDPN asdetermined is 20% or more up-regulated in cells relative to thatdetermined in benign epithelial cells of the same specimen, the animalsubject is identified as being likely to have glioblastoma.

The more different proteins of the group comprising BCA, CLIP2, DTNA,NLGNAX, NR2E1, NRCAM, and PDPN are up-regulated the higher thepossibility of the animal subject is identified as being likely to haveglioblastoma.

In one embodiment, the determination of the level of BCA, CLIP2, DTNA,NLGNAX, NR2E1, NRCAM or PDPN is performed in situ. In anotherembodiment, the determination of the level of BCA, CLIP2, DTNA, NLGNAX,NR2E1, NRCAM or PDPN is performed in vitro. In still another embodiment,the determination of the level of BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAMor PDPN is performed in vivo. In a preferred embodiment, thedetermination of the level of BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM orPDPN is performed by Laser Capture Microscopy coupled with a Westernblot.

In a particular embodiment, the determination of the level of BCA,CLIP2, DTNA, NLGNAX, NR2E1, NRCAM or PDPN is performed with an antibodyspecific for BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM or PDPN. In anotherembodiment, the determination of the level of BCA, CLIP2, DTNA, NLGNAX,NR2E1, NRCAM or PDPN is performed by PCR with a primer specific for anmRNA encoding BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM or PDPN. In stillanother embodiment, the determination of the level of BCA, CLIP2, DTNA,NLGNAX, NR2E1, NRCAM or PDPN is performed with a nucleotide probespecific for an mRNA encoding BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM orPDPN. In one embodiment, the determination of the level of BCA, CLIP2,DTNA, NLGNAX, NR2E1, NRCAM or PDPN is performed using a Northern blot.In another embodiment, the determination of the level of BCA, CLIP2,DTNA, NLGNAX, NR2E1, NRCAM or PDPN is achieved using a ribonucleaseprotection assay. In other embodiments, immunological tests such asenzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), andWestern blots may be used to detect BCA, CLIP2, DTNA, NLGNAX, NR2E1,NRCAM, and PDPN polypeptides in a body fluid sample (such as blood,serum, sputum, urine, or peritoneal fluid). Biopsies, tissue samples,and cell samples (such as ovaries, lymph nodes, ovarian surfaceepithelial cell scrapings, lung biopsies, liver biopsies, and any fluidsample containing cells (such as peritoneal fluid, sputum, and pleuraleffusions) may be tested by disaggregating and/or solubilizing thetissue or cell sample and subjecting it to an immunoassay forpolypeptide detection, such as ELISA, RIA, or Western blotting. Suchcell or tissue samples may also be analyzed by nucleic acid-basedmethods, e.g., reverse transcription-polymerase chain reaction (RT-PCR)amplification, Northern hybridization, or slot- or dot-blotting. Tovisualize the distribution of tumor cells within a tissue sample,diagnostic tests that preserve the tissue structure of a sample, e.g.,immunohistological staining, in situ RNA hybridization, or in situRT-PCR may be employed to detect glioblastoma marker polypeptide ormRNA, respectively. For in vivo localization of tumor masses, imagingtests such as magnetic resonance imaging (MRI) may be employed byintroducing into the subject an antibody that specifically binds a BCA,CLIP2, DTNA, NLGNAX, NR2E1, NRCAM or PDPN polypeptide (particularly acell surface-localized polypeptide), wherein the antibody is conjugatedor otherwise coupled to a paramagnetic tracer (or other appropriatedetectable moiety, depending upon the imaging method used);alternatively, localization of an unlabeled tumor marker-specificantibody may be detected using a secondary antibody coupled to adetectable moiety.

In addition, the present invention further provides chimeric/fusionproteins/peptides comprising the BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAMand/or PDPN polypeptides, and fragments thereof, including functional,proteolytic and antigenic fragments.

The fusion partner or sections of a hybrid molecule suitably provideepitopes that stimulate CD4⁺ T-cells. CD4⁺ stimulating epitopes are wellknown in the art and include those identified in tetanus toxoid. In afurther preferred embodiment the peptide is a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii). In one embodiment the peptideof the invention is a truncated human protein or a fusion protein of aprotein fragment and another polypeptide portion provided that the humanportion includes one or more inventive amino acid sequences.

Antibodies to the BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM or PDPNpolypeptides, to the chimeric/fusion proteins comprising the BCA, CLIP2,DTNA, NLGNAX, NR2E1, NRCAM or PDPN polypeptides, as well as to thefragments of the BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM or PDPNpolypeptides, including proteolytic, and antigenic fragments, and to thechimeric/fusion proteins/peptides comprising these fragments are alsopart of the present invention. In addition, methods of using suchantibodies for the prognosis of cancer, and glioblastoma in particular,are also part of the present invention.

The antibodies of the present invention can be polyclonal antibodies,monoclonal antibodies and/or chimeric antibodies. Immortal cell linesthat produce a monoclonal antibody of the present invention are alsopart of the present invention.

One of ordinary skill in the art will understand that in some instances,higher expression of BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM or PDPN as atumor marker gene will indicate a worse prognosis for a subject havingglioblastoma. For example, relatively higher levels BCA, CLIP2, DTNA,NLGNAX, NR2E1, NRCAM or PDPN expression may indicate a relative largeprimary tumor, a higher tumor burden (e.g., more metastases), or arelatively more malignant tumor phenotype.

The more the overexpression of the different proteins of the groupcomprising BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and PDPN is different,the worse is the prognosis for a patient.

The diagnostic and prognostic methods of the invention involve usingknown methods, e.g., antibody-based methods to detect BCA, CLIP2, DTNA,NLGNAX, NR2E1, NRCAM and PDPN polypeptides and nucleic acidhybridization- and/or amplification-based methods to detect BCA, CLIP2,DTNA, NLGNAX, NR2E1, NRCAM and/or PDPN mRNA.

In addition, since rapid tumor cell destruction often results inautoantibody generation, the glioblastoma tumor markers of the inventionmay be used in serological assays (e.g., an ELISA test of a subject'sserum) to detect autoantibodies against BCA, CLIP2, DTNA, NLGNAX, NR2E1,NRCAM or PDPN in a subject. BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM andPDPN polypeptide-specific autoantibody levels that are at least about3-fold higher (and preferably at least 5-fold or 7-fold higher, mostpreferably at least 10-fold or 20-fold higher) than in a control sampleare indicative of glioblastoma.

Cell-surface localized, intracellular, and secreted BCA, CLIP2, DTNA,NLGNAX, NR2E1, NRCAM and PDPN polypeptides may all be employed foranalysis of biopsies, e.g., tissue or cell samples (including cellsobtained from liquid samples such as peritoneal cavity fluid) toidentify a tissue or cell biopsy as containing glioblastoma cells. Abiopsy may be analyzed as an intact tissue or as a whole-cell sample, orthe tissue or cell sample may be disaggregated and/or solubilized asnecessary for the particular type of diagnostic test to be used. Forexample, biopsies or samples may be subjected to whole-tissue orwhole-cell analysis of BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and PDPNpolypeptide or mRNA levels in situ, e.g., using immunohistochemistry, insitu mRNA hybridization, or in situ RT-PCR. The skilled artisan willknow how to process tissues or cells for analysis of polypeptide or mRNAlevels using immunological methods such as ELISA, immunoblotting, orequivalent methods, or analysis of mRNA levels by nucleic acid-basedanalytical methods such as RT-PCR, Northern hybridization, or slot- ordot-blotting.

Kits for Measuring Expression Levels of BCA, CLIP2, DTNA, NLGNAX, NR2E1,NRCAM and PDPN

The present invention provides kits for detecting an increasedexpression level of BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and PDPN as aglioblastoma marker gene in a subject. A kit for detecting glioblastomamarker polypeptide preferably contains an antibody that specificallybinds a chosen glioblastoma marker polypeptide. A kit for detectingglioblastoma marker mRNA preferably contains one or more nucleic acids(e.g., one or more oligonucleotide primers or probes, DNA probes, RNAprobes, or templates for generating RNA probes) that specificallyhybridize with BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and PDPN mRNA.

Particularly, the antibody-based kit can be used to detect the presenceof, and/or measure the level of, a BCA, CLIP2, DTNA, NLGNAX, NR2E1,NRCAM and/or PDPN polypeptide that is specifically bound by the antibodyor an immunoreactive fragment thereof. The kit can include an antibodyreactive with the antigen and a reagent for detecting a reaction of theantibody with the antigen. Such a kit can be an ELISA kit and cancontain a control (e.g., a specified amount of a particular glioblastomamarker polypeptide), primary and secondary antibodies when appropriate,and any other necessary reagents such as detectable moieties, enzymesubstrates and color reagents as described above. The diagnostic kitcan, alternatively, be an immunoblot kit generally comprising thecomponents and reagents described herein.

A nucleic acid-based kit can be used to detect and/or measure theexpression level of BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and PDPN bydetecting and/or measuring the amount of BCA, CLIP2, DTNA, NLGNAX,NR2E1, NRCAM and PDPN mRNA in a sample, such as a tissue or cell biopsy.For example, an RT-PCR kit for detection of elevated expression of BCA,CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and PDPN preferably containsoligonucleotide primers sufficient to perform reverse transcription ofglioblastoma marker mRNA to cDNA and PCR amplification of glioblastomamarker cDNA, and will preferably also contain control PCR templatemolecules and primers to perform appropriate negative and positivecontrols, and internal controls for quantization. One of ordinary skillin the art will understand how to select the appropriate primers toperform the reverse transcription and PCR reactions, and the appropriatecontrol reactions to be performed. Such guidance is found, for example,in F. Ausubel et al., Current Protocols in Molecular Biology, John Wiley& Sons, New York, N.Y., 1997. Numerous variations of RT-PCR are known inthe art. Targeted Delivery of immunotoxins to BCA, CLIP2, DTNA, NLGNAX,NR2E1, NRCAM and PDPN can be employed as therapeutic targets for thetreatment or prevention of glioblastoma. For example, an antibodymolecule that specifically binds a cell surface-localized BCA, CLIP2,DTNA, NLGNAX, NR2E1, NRCAM and PDPN polypeptide can be conjugated to aradioisotope or other toxic compound. Antibody conjugates areadministered to the subject so that the binding of the antibody to itscognate glioblastoma polypeptide results in the targeted delivery of thetherapeutic compound to glioblastoma cells, thereby treating an ovariancancer.

The therapeutic moiety can be a toxin, radioisotope, drug, chemical, ora protein (see, e.g., Bera et al. “Pharmacokinetics and antitumoractivity of a bivalent disulfide-stabilized Fv immunotoxin with improvedantigen binding to erbB2” Cancer Res. 59:4018-4022 (1999)). For example,the antibody can be linked or conjugated to a bacterial toxin (e.g.,diptheria toxin, pseudomonas exotoxin A, cholera toxin) or plant toxin(e.g., ricin toxin) for targeted delivery of the toxin to a cellexpressing BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and PDPN. Thisimmunotoxin can be delivered to a cell and upon binding the cellsurface-localized glioblastoma marker polypeptide, the toxin conjugatedto the glioblastoma marker-specific antibody will be delivered to thecell.

Yet another aspect of the present invention relates to an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen (in thefollowing also designate as “complex-specific antibody”). Yet anotheraspect of the present invention then relates to a method of producingsaid antibody specifically binding to a human major histocompatibilitycomplex (MHC) class I or II being complexed with a HLA-restrictedantigen, the method comprising: immunizing a genetically engineerednon-human mammal comprising cells expressing said human majorhistocompatibility complex (MHC) class I or II with a soluble form of aMHC class I or II molecule being complexed with said HLA-restrictedantigen; isolating mRNA molecules from antibody producing cells of saidnon-human mammal; producing a phage display library displaying proteinmolecules encoded by said mRNA molecules; and isolating at least onephage from said phage display library, said at least one phagedisplaying said antibody specifically bindable to said human majorhistocompatibility complex (MHC) class I or II being complexed with saidHLA-restricted antigen. Respective methods for producing such antibodiesand single chain class I major histocompatibility complexes, as well asother tools for the production of these antibodies are disclosed in WO03/068201, WO 2004/084798, WO 01/72768, WO 03/070752, and Cohen C J,Denkberg G, Lev A, Epel M, Reiter Y. Recombinant antibodies withMHC-restricted, peptide-specific, T-cell receptor-like specificity: newtools to study antigen presentation and TCR-peptide-MHC interactions. JMol Recognit. 2003 September-October; 16(5):324-32.; Denkberg G, Lev A,Eisenbach L, Benhar I, Reiter Y. Selective targeting of melanoma andAPCs using a recombinant antibody with TCR-like specificity directedtoward a melanoma differentiation antigen. J Immunol. 2003 Sep. 1;171(5):2197-207; and Cohen CJ, Sarig O, Yamano Y, Tomaru U, Jacobson S,Reiter Y. Direct phenotypic analysis of human MHC class I antigenpresentation: visualization, quantitation, and in situ detection ofhuman viral epitopes using peptide-specific, MHC-restricted humanrecombinant antibodies. J Immunol. 2003 Apr. 15; 170(8):4349-61, whichfor the purposes of the present invention are all explicitlyincorporated by reference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isregarded as “specific” in the context of the present invention.

The term “antibody” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of those immunoglobulin molecules and humanizedversions of immunoglobulin molecules, so long as they exhibit any of thedesired properties (e.g., being a complex-specific antibody as above,delivery of a toxin to a cancer cell expressing an cancer preferred aglioblastoma marker gene at an increased level, and/or inhibiting theactivity of an cancer marker polypeptide, such as survivin) describedherein.

In addition, for any BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and PDPNpolypeptide for which there is a specific ligand (e.g., a ligand thatbinds a cell surface-localized protein), the ligand can be used in placeof an antibody to target a toxic compound to a glioblastoma cell, asdescribed above.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length glioblastoma marker polypeptides or fragmentsthereof may be used to generate the antibodies of the invention. Apolypeptide to be used for generating an antibody of the invention maybe partially or fully purified from a natural source, or may be producedusing recombinant DNA techniques. For example, a cDNA encoding a BCA,CLIP2, DTNA, NLGNAX, NR2E1, NRCAM or PDPN polypeptide, or a fragmentthereof, can be expressed in prokaryotic cells (e.g., bacteria) oreukaryotic cells (e.g., yeast, insect, or mammalian cells), after whichthe recombinant protein can be purified and used to generate amonoclonal or polyclonal antibody preparation that specifically bind theglioblastoma marker polypeptide used to generate the antibody.

One of skill in the art will know that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistiy,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1988). For example, the antibodies may be tested in ELISA assays,Western blots, immunohistochemical staining of formalin-fixedglioblastomas or frozen tissue sections. After their initial in vitrocharacterization, antibodies intended for therapeutic or in vivodiagnostic use are tested according to known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348, published 22 Dec. 1994,and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fe fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross-linking antigen.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the nonmodified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. Antibodies in Human Diagnosis and Therapy, Haber etal, eds. Raven Press, New York (1977) pp. 365-389. A typical dailydosage of the antibody used alone might range from about 1 (μg/kg to upto 100 mg/kg of body weight or more per day, depending on the factorsmentioned above. Following administration of an antibody for treatingglioblastoma, the efficacy of the therapeutic antibody can be assessedin various ways well known to the skilled practitioner. For instance,the size, number, and/or distribution of glioblastoma in a subjectreceiving treatment may be monitored using standard tumor imagingtechniques. A therapeutically-administered antibody that arrests tumorgrowth, results in tumor shrinkage, and/or prevents the development ofnew tumors, compared to the disease course that would occurs in theabsence of antibody administration, is an efficacious antibody fortreatment of glioblastoma.

Because the glioblastoma tumor marker BCA, CLIP2, DTNA, NLGNAX, NR2E1,NRCAM and PDPN of the invention are highly expressed in glioblastomacells and is expressed at extremely low levels in normal cells,inhibition of BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM or PDPN expressionor polypeptide activity may be integrated into any therapeutic strategyfor treating or preventing glioblastoma.

The principle of antisense therapy is based on the hypothesis thatsequence-specific suppression of gene expression (via transcription ortranslation) may be achieved by intra-cellular hybridization betweengenomic DNA or mRNA and a complementary antisense species. The formationof such a hybrid nucleic acid duplex interferes with transcription ofthe target tumor antigen-encoding genomic DNA, orprocessing/transport/translation and/or stability of the target tumorantigen mRNA.

Antisense nucleic acids can be delivered by a variety of approaches. Forexample, antisense oligonucleotides or anti-sense RNA can be directlyadministered (e.g., by intravenous injection) to a subject in a formthat allows uptake into tumor cells. Alternatively, viral or plasmidvectors that encode antisense RNA (or RNA fragments) can be introducedinto cells in vivo. Antisense effects can also be induced by sensesequences; however, the extent of phenotypic changes is highly variable.Phenotypic changes induced by effective antisense therapy are assessedaccording to changes in, e.g., target mRNA levels, target proteinlevels, and/or target protein activity levels.

In a specific example, inhibition of glioblastoma marker function byantisense gene therapy may be accomplished by direct administration ofantisense glioblastoma marker RNA to a subject. The antisense tumormarker RNA may be produced and isolated by any standard technique, butis most readily produced by in vitro transcription using an antisensetumor marker cDNA under the control of a high efficiency promoter (e.g.,the T7 promoter). Administration of anti-sense tumor marker RNA to cellscan be carried out by any of the methods for direct nucleic acidadministration described below.

An alternative strategy for inhibiting BCA, CLIP2, DTNA, NLGNAX, NR2E1,NRCAM and/or PDPN function using gene therapy involves intracellularexpression of an anti-BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM or PDPNantibody or a portion of an anti-BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAMor PDPN antibody. For example, the gene (or gene fragment) encoding amonoclonal antibody that specifically binds to a BCA, CLIP2, DTNA,NLGNAX, NR2E1, NRCAM or PDPN polypeptide and inhibits its biologicalactivity is placed under the transcriptional control of a specific(e.g., tissue- or tumor-specific) gene regulatory sequence, within anucleic acid expression vector. The vector is then administered to thesubject such that it is taken up by glioblastoma cells or other cells,which then secrete the anti-BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM orPDPN antibody and thereby block biological activity of the BCA, CLIP2,DTNA, NLGNAX, NR2E1, NRCAM and PDPN polypeptide. Preferably, the BCA,CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and PDPN polypeptides are present atthe extracellular surface of glioblastoma cells.

In the methods described above, which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the nucleic acids of the presentinvention can be in the form of naked DNA or the nucleic acids can be ina vector for delivering the nucleic acids to the cells for inhibition ofglioblastoma marker protein expression. The vector can be a commerciallyavailable preparation, such as an adenovirus vector (QuantumBiotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleicacid or vector to cells can be via a variety of mechanisms. As oneexample, delivery can be via a liposome, using commercially availableliposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-25 BRL,Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) andTRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as otherliposomes developed according to procedures standard in the art. Inaddition, the nucleic acid or vector of this invention can be deliveredin vivo by electroporation, the technology for which is available fromGenetronics, Inc. (San Diego, Calif.) as well as by means of aSONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system that can package a recombinant retroviralgenome. The recombinant retrovirus can then be used to infect andthereby deliver to the infected cells antisense nucleic acid thatinhibits expression of BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM or PDPN.The exact method of introducing the altered nucleic acid into mammaliancells is, of course, not limited to the use of retroviral vectors. Othertechniques are widely available for this procedure including the use ofadenoviral vectors, adeno-associated viral (AAV) vectors, lentiviralvectors, pseudotyped retroviral vectors. Physical transductiontechniques can also be used, such as liposome delivery andreceptor-mediated and other endocytosis mechanisms. This invention canbe used in conjunction with any of these or other commonly used genetransfer methods.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P, ³⁵S) so that the tumor can be localizedusing immunoscintiography. In one embodiment, antibodies or fragmentsthereof bind to the extracellular domains of two or more BCA, CLIP2,DTNA, NLGNAX, NR2E1, NRCAM and PDPN targets and the affinity value (Kd)is less than 1×10 μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect the BCA,CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and PDPN proteins express in situ orin vitro.

The present invention in another preferred aspect thereof provides apeptide comprising a sequence that is selected from the group of SEQ IDNO:1 to SEQ ID NO:30 or a variant thereof which is 85% homologous to SEQID NO:1 to SEQ ID NO:30 or a variant thereof that will induce T cellscross-reacting with said peptide.

In a preferred embodiment the peptide is selected from a group of thepeptides comprising a sequence that is selected from the group of SEQ IDNO:1 to SEQ ID NO:24 or a variant thereof which is 85% homologous to SEQID NO:1 to SEQ ID NO:24 or a variant thereof that will induce T cellscross-reacting with said peptide.

The peptides of the invention have the ability to bind to a molecule ofthe human major histocompatibility complex (MHC) class-I or -II.

In the present invention, the term “homologous” refers to the degree ofidentity between sequences of two amino acid sequences, i.e. peptide orpolypeptide sequences. The aforementioned “homology” is determined bycomparing two sequences aligned under optimal conditions over thesequences to be compared. The sequences to be compared herein may havean addition or deletion (for example, gap and the like) in the optimumalignment of the two sequences. Such a sequence homology can becalculated by creating an alignment using, for example, the ClustalWalgorithm (Nucleic Acid Res., 22(22): 4673 4680 (1994). Commonlyavailable sequence analysis software, more specifically, Vector NTI,GENETYX or analysis tools provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Fong et al., 2001); (Zaremba et al., 1997;Colombetti et al., 2006; Appay et al., 2006).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in SEQ ID NO:1 to 30. For example, a peptide may bemodified so that it at least maintains, if not improves, the ability tointeract with and bind to the binding groove of a suitable MHC molecule,such as HLA-A*02 or -DR, and in that way it at least maintains, if notimproves, the ability to bind to the TCR of activated CTL. These CTL cansubsequently cross-react with cells and kill cells that express apolypeptide which contains the natural amino acid sequence of thecognate peptide as defined in the aspects of the invention. As can bederived from the scientific literature (Rammensee et al., 1997) anddatabases (Rammensee et al., 1999), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ IDNO:1 to 30, by maintaining the known anchor residues, and would be ableto determine whether such variants maintain the ability to bind MHCclass I or II molecules. The variants of the present invention retainthe ability to bind to the TCR of activated CTL, which can subsequentlycross-react with- and kill cells that express a polypeptide containingthe natural amino acid sequence of the cognate peptide as defined in theaspects of the invention.

Those amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith another amino acid whose incorporation does not substantiallyaffect T-cell reactivity and does not eliminate binding to the relevantMHC. Thus, apart from the proviso given, the peptide of the inventionmay be any peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 2 Variants and motif of the peptides according to SEQ ID NO: 1 to25 Position 1 2 3 4 5 6 7 8 9 CSP-001 Peptide Code T M L A R L A S A SEQID NO: 25 Variants L L L E K I A G I I A E L Y P K Y F F T Y T H K P N MM F Y S V V R Position 1 2 3 4 5 6 7 8 9 ACS-001 Peptide Code K I M E RI Q E V SEQ ID NO: 3 Variants M L L L K I A G I A L Y P K L Y F F T Y TH P N M F Y S V V R Position 1 2 3 4 5 6 7 8 9 BCA-001 Peptide Code F LG D P P E K L SEQ ID NO: 4 Variants M E I A G I I A E L Y P K L Y F T YT H K P N M M F Y S V V R Position 1 2 3 4 5 6 7 8 9 BCA-002 PeptideCode A L W A W P S E L SEQ ID NO: 5 Variants M E K I A G I I A L Y P K LY F F T Y T H K P N M M F Y S V V R Position 1 2 3 4 5 6 7 8 9CHI3L1-010 Peptide Code T L Y G M L N T L SEQ ID NO: 6 Variants M E K IA I I A E L P K Y F F T Y T H K P N M M F Y S V V R Position 1 2 3 4 5 67 8 9 CLIP2-001 Peptide Code S L N E L R V L L SEQ ID NO: 7 Variants M KI A G I I A E L Y P K L Y F F T Y T H K P N M M F Y S V V R Position 1 23 4 5 6 7 8 9 SLCO1C1-001 Peptide Code Y L I A G I I S L SEQ ID NO: 2Variants M E K I A G I A E L Y P K L Y F F T Y T H K P N M M F S V V RPosition 1 2 3 4 5 6 7 8 9 DTNA-001 Peptide Code K L Q D E A Y Q V SEQID NO: 8 Variants M L L E K I A G I I A E L Y P K L F F T Y T H P N M MF Y S V V R Position 1 2 3 4 5 6 7 8 9 EGFR-007 Peptide Code A L A V L SN Y D SEQ ID NO: 9 Variants M L L E K I A G I I A E L Y P K L Y F F T YT H K P N M M F Y S V V R Position 1 2 3 4 5 6 7 8 9 FABP7-001 PeptideCode L T F G D V V A V SEQ ID NO: 10 Variants M L L L E K I A I I A E YP K L Y F T Y T H K P N M M F Y S V V R Position 1 2 3 4 5 6 7 8 9GFAP-001 Peptide Code N L A Q D L A T V SEQ ID NO: 11 Variants M L L E KI G I I E L Y P K Y F F T Y T H K P N M M F Y S V V R Position 1 2 3 4 56 7 8 9 GPR56-002 Peptide Code F L L S E P V A L SEQ ID NO: 12 VariantsM E K I A G I I A E L Y P K L Y F T Y T H K P N M M F Y S V V R Position1 2 3 4 5 6 7 8 9 GRI-001 Peptide Code N I L E Q I V S V SEQ ID NO: 13Variants M L L L E K I A G I I A E L Y P K L Y F F T Y T H K P N M M F YS V V R Position 1 2 3 4 5 6 7 8 9 IGF2BP3-001 Peptide Code K I Q E I LT Q V SEQ ID NO: 14 Variants M L L L K I A G I A E L Y P K Y F F T Y T HP N M M F Y S V V R Position 1 2 3 4 5 6 7 8 9 MLC-001 Peptide Code S VV E V I A G I SEQ ID NO: 15 Variants M L L L K I A G I E L Y P K L Y F FT Y T H K P N M M F Y S V R Position 1 2 3 4 5 6 7 8 9 NES-001 PeptideCode G L Q S Q I A Q V SEQ ID NO: 16 Variants M L L E K I A G I E L Y PK L Y F F T Y T H K P N M M F Y S V V R Position 1 2 3 4 5 6 7 8 9NES-002 Peptide Code S L Q E N L E S L SEQ ID NO: 17 Variants M K I A GI I A E L Y P K Y F F T Y T H K P M M F Y S V V R Position 1 2 3 4 5 6 78 9 NES-003 Peptide Code F L F P G T E N Q SEQ ID NO: 18 Variants M L LE K I A G I I A E L Y K L Y T Y H K P N M M F Y S V V R Position 1 2 3 45 6 7 8 9 NES-004 Peptide Code N L A E E L E G V SEQ ID NO: 19 VariantsM L L K I G I I A E L Y P K Y F F T Y T H K P N M M F Y S V V R Position1 2 3 4 5 6 7 8 9 NLGN4X-001 Peptide Code N L D T L M T Y V SEQ ID NO: 1Variants M L L E K I A G I I A E L Y P K L Y F F Y T H K P N M M F Y S VV R Position 1 2 3 4 5 6 7 8 9 NR2E1-001 Peptide Code K I I S E I Q A LSEQ ID NO: 20 Variants M L E K I A G I A E L Y P K L Y F F T Y T H P N MM F Y S V V R Position 1 2 3 4 5 6 7 8 9 NRCAM-001 Peptide Code G L W HH Q T E V SEQ ID NO: 21 Variants M L L E K I A G I I A E L Y P K L Y F FT Y T H K P N M M F Y S V V R Position 1 2 3 4 5 6 7 8 9 PDPN-001Peptide Code T L V G I I V G V SEQ ID NO: 22 Variants M L L E K I A A EL Y P K L Y F F T Y T H K P N M M F Y S V V R Position 1 2 3 4 5 6 7 8 9TNC-001 Peptide Code A M T Q L L A G V SEQ ID NO: 23 Variants L L L E KI A G I I E L Y P K Y F F T Y T H K P N M M F Y S V V R Position 1 2 3 45 6 7 8 9 TNC-002 Peptide Code Q L L A G V F L A SEQ ID NO: 24 VariantsM L L E K I A G I I A E L Y P K L Y F F T Y T H K P N M M F Y S V V R

It is furthermore known for MHC-class II-presented peptides that thesepeptides are composed of a “core sequence” having an amino acid sequencefitting to a certain HLA-allele-specific motif and, optionally, N-and/or C-terminal extensions that do not interfere with the function ofthe core sequence (i.e. are deemed as irrelevant for the interaction ofthe peptide and all or a subset of T cell clones recognizing the naturalcounterpart). The N- and/or C-terminal extensions can, for example, bebetween 1 to 10 amino acids in length, respectively. These peptides canbe used either directly in order to load MHC class II molecules or thesequence can be cloned into the vectors according to the descriptionherein below. As these peptides constitute the final product of theprocessing of larger peptides within the cell, longer peptides can beused as well. The peptides of the invention may be of any size, buttypically they may be less than 100,000 in molecular weight, preferablyless than 50,000, more preferably less than 10,000 and typically about5,000. In terms of the number of amino acid residues, the peptides ofthe invention may have fewer than 1,000 residues, preferably fewer than500 residues, more preferably fewer than 100, more preferably fewer than100 and most preferably between 30 and 8 residues. Accordingly, thepresent invention also provides peptides and variants thereof whereinsaid peptide or variant has an overall length of between 8 and 100,preferably between 8 and 30, and most preferred between 8 and 16, namely8, 9, 10, 11, 12, 13, 14, 15, 16 amino acids.

Longer peptides may also be suitable, 9-mer or 10-mer peptides asdescribed in the above Table 2 are preferred for MHC class I-peptides,while 12- to 15-mers are preferred for MHC class II peptides.

For MHC class II restricted peptides, several different peptides withthe same core sequence may be presented in the MHC molecule. As theinteraction with the recognizing T (helper) cell is defined by a coresequence of 9 to 11 amino acids, several length variants may berecognized by the same T (helper) cell clone. Thus, several differentlengths variants of a core binding sequence may be used for directloading of MHC class II molecules without the nee for further processingand trimming at the N- or C-terminal ends. Correspondingly, naturallyoccurring or artificial variants that induce T cells cross-reacting witha peptide of the invention are often length variants.

If a peptide that is longer than around 12 amino acid residues is useddirectly to bind to a MHC class II molecule, it is preferred that theresidues that flank the core HLA binding region are residues that do notsubstantially affect the ability of the peptide to bind specifically tothe binding groove of the MHC class II molecule or to present thepeptide to the T (-helper) cell. However, as already indicated above, itwill be appreciated that larger peptides may be used, e.g. when encodedby a polynucleotide, since these larger peptides may be fragmented bysuitable antigen-presenting cells. However, in same cases it has beenshown that the core sequence flanking regions can influence the peptidebinding to MHC class II molecule or the interaction of the dimericMHC:peptide complex with the TCR in both directions compared to areference peptide with the same core sequence. Intramolecular tertiarystructures within the peptide (e.g. loop formation) normally decreasethe affinities to the MHC or TCR. Intermolecular interactions of theflanking regions with parts of the MHC or TCR beside the peptide bindinggrooves may stabilize the interaction. These changes in affinity canhave a dramatic influence on the potential of a MHC class II peptide toinduce T (helper) cell responses.

It is also possible that MHC class I epitopes, although usually between8-10 amino acids long, are generated by peptide processing from longerpeptides or proteins that include the actual epitope. It is preferredthat the residues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

Preferred are therefore peptides with a core sequence selected from agroup consisting of SEQ ID No 1 to SEQ ID No 30 with extensions of 1 to10 amino acids on the C-terminal and/or the N-terminal, more preferredthe overall number of these flanking amino acids is 1 to 12, morepreferred 1 to 10, more preferred 1 to 8, more preferred 1 to 6, morepreferred 1 to 4 and even more preferred 1 to 2, wherein the flankingamino acids can be distributed in any ratio to the C-terminus and theN-terminus (for example all flanking amino acids can be added to oneterminus, or the amino acids can be added equally to both termini or inany other ratio), provided that the peptide is still able to bind to anHLA molecule in the same way as said peptide according to any of the SEQID No 1 to SEQ ID No 30.

The flanking amino acids can also reduce the speed of peptidedegradation in vivo so that the amount of the actual peptide availableto the CTLs is higher compared to the peptide without flanking aminoacids, thus acting as a prodrug.

Accordingly, the present invention also provides peptides and variantsof MHC class I epitopes wherein the peptide or variant has an overalllength of between 8 and 100, preferably between 8 and 30, and mostpreferred between 8 and 16, namely 8, 9, 10, 11, 12, 13, 14, 15, 16amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art,for example those described in the literature for different MHC class IIalleles (e.g. (Vogt et al., 1994; Malcherek et al., 1994; Manici et al.,1999; Hammer et al., 1995; Tompkins et al., 1993; Boyton et al., 1998)).

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 30.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID No. 1 to SEQ ID No. 30 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is afusion protein which comprises, for example, the 80 N-terminal aminoacids of the HLA-DR antigen-associated invariant chain (p33, in thefollowing “Ii”) as derived from the NCBI, GenBank Accession-numberX00497 (Strubin, M. et al 1984).

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) J. Immunol. 159,3230-3237, incorporated herein by reference. This approach involvesmaking pseudopeptides containing changes involving the backbone, and notthe orientation of side chains. Meziere et al (1997) show that for MHCbinding and T helper cell responses, these pseudopeptides are useful.Retro-inverse peptides, which contain NH—CO bonds instead of CO—NHpeptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2005, which is incorporatedherein by reference. Chemical modification of amino acids includes butis not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulfonic acid (TNBS), amidemodification of carboxyl groups and sulfhydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulfides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley & Sons NY 1995-2000) for more extensivemethodology relating to chemical modification of proteins.

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethyleneglycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Generally,peptides and variants (at least those containing peptide linkagesbetween amino acid residues) may be synthesized by the Fmoc-polyamidemode of solid-phase peptide synthesis as disclosed by Lu et al (1981)and references therein. Temporary N-amino group protection is affordedby the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage ofthis highly base-labile protecting group is done using 20% piperidine inN, N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/lhydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used include ethanedithiol, phenol, anisole andwater, the exact choice depending on the constituent amino acids of thepeptide being synthesized. Also a combination of solid phase andsolution phase methodologies for the synthesis of peptides is possible(see, for example, Bruckdorfer et al. 2004, and the references as citedtherein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilisation of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, CNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc, New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by (Saikiet al (1988)). This method may be used for introducing the DNA into asuitable vector, for example by engineering in suitable restrictionsites, or it may be used to modify the DNA in other useful ways as isknown in the art. If viral vectors are used, pox- or adenovirus vectorsare preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed in U.S. Pat. Nos. 4,440,859, 4,530,901, 4,582,800, 4,677,063,4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3xFLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB 1 (derivative of pBR322) origin forreplication in bacterial cells, the beta-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the fl origin. Vectorscontaining the preprotrypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl.Acad. Sci. USA 69, 2110, and Sambrook et al (1989) Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. Transformation of yeast cells is described in Sherman et al (1986)Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y.The method of Beggs (1978) Nature 275,104-109 is also useful. Withregard to vertebrate cells, reagents useful in transfecting such cells,for example calcium phosphate and DEAE-dextran or liposome formulations,are available from Stratagene Cloning Systems, or Life TechnologiesInc., Gaithersburg, Md. 20877, USA. Electroporation is also useful fortransforming and/or transfecting cells and is well known in the art fortransforming yeast cell, bacterial cells, insect cells and vertebratecells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) are currently under investigation for the treatment of prostatecancer (Sipuleucel-T) (Small E J et al 2006; Rini et al 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Doses of this range were successfully used inprevious trials (Brunsvig et al 2006; Staehler et al 2007).

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human class I or II MHC molecules expressed onthe surface of a suitable antigen-presenting cell for a period of timesufficient to activate the T cell in an antigen specific manner, whereinthe antigen is a peptide according to the invention. Preferably asufficient amount of the antigen is used with an antigen-presentingcell.

In the case of a MHC class II epitope being used as an antigen, the Tcells are CD4-positive helper cells, preferably of T_(H1)-type. The MHCclass II molecules may be expressed on the surface of any suitable cell.Preferably the cell does not naturally express MHC class II molecules(in which case the cell has been transfected in order to express such amolecule). Alternatively, if the cell naturally expresses MHC class IImolecules, it is preferred that it is defective in theantigen-processing or antigen-presenting pathways. In this way, it ispossible for the cell expressing the MHC class II molecule to becompletely loaded with a chosen peptide antigen before activating the Tcell.

The antigen-presenting cell (or stimulator cell) typically has MHC classII molecules on its surface and preferably is itself substantiallyincapable of loading said MHC class II molecule with the selectedantigen. The MHC class II molecule may readily be loaded with theselected antigen in vitro.

Preferably, the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is theTransporter associated with Antigen Processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Karre et al 1985.

Preferably, the host cell before transfection expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class II molecules and of the costimulatormolecules are publicly available from the GenBank and EMBL databases.

Similarly, in case of a MHC class I epitope being used as an antigen,the T cells are CD8-positive CTLs.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 30 or a variant aminoacid sequence thereof.

A number of other methods may be used for generating CTL in vitro. Forexample, the methods described in Peoples et al (1995) and Kawakami etal (1992) use autologous tumor-infiltrating lymphocytes in thegeneration of CTL. Plebanski et al (1995) makes use of autologousperipheral blood lymphocytes (PLBs) in the preparation of CTL. Jochmuset al (1997) describes the production of autologous CTL by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus. Hill et al (1995) and Jerome et al (1993) make use ofB cells in the production of autologous CTL. In addition, macrophagespulsed with peptide or polypeptide, or infected with recombinant virus,may be used in the preparation of autologous CTL. S. Walter et al. 2003describe the in vitro priming of T cells by using artificial antigenpresenting cells (aAPCs), which is also a suitable way for generating Tcells against the peptide of choice. In this study, aAPCs were generatedby the coupling of preformed MHC:peptide complexes to the surface ofpolystyrene particles (microbeads) by biotin:streptavidin biochemistry.This system permits the exact control of the MHC density on aAPCs, whichallows to selectively elicit high- or low-avidity antigen-specific Tcell responses with high efficiency from blood samples. Apart fromMHC:peptide complexes, aAPCs should carry other proteins withco-stimulatory activity like anti-CD28 antibodies coupled to theirsurface. Furthermore such aAPC-based systems often require the additionof appropriate soluble factors, e.g. cytokines like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328. For example, in additionto Drosophila cells and T2 cells, other cells may be used to presentantigens such as CHO cells, baculovirus-infected insect cells, bacteria,yeast, vaccinia-infected target cells. In addition plant viruses may beused (see, for example, Porta et al (1994)) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to 30.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease which can be readily tested for, anddetected.

In vivo, the target cells for the CD4-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to normal levels of expression or that thegene is silent in the tissue from which the tumor is derived but in thetumor it is expressed. By “over-expressed” the inventors mean that thepolypeptide is present at a level at least 1.2-fold of that present innormal tissue; preferably at least 2-fold, and more preferably at least5-fold or 10-fold the level present in normal tissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art and can be found, e.g. in (Rosenberg et al., 1987; Rosenberget al., 1988; Dudley et al., 2002; Yee et al., 2002; Dudley et al.,2005); reviewed in (Gattinoni et al., 2006) and (Morgan et al., 2006).

Any molecule of the invention, i.e. the peptide, nucleic acid,expression vector, cell, activated CTL, T-cell receptor or the nucleicacid encoding it is useful for the treatment of disorders, characterizedby cells escaping an immune response. Therefore any molecule of thepresent invention may be used as medicament or in the manufacture of amedicament. The molecule may be used by itself or combined with othermolecule(s) of the invention or (a) known molecule(s).

Preferably, the medicament of the present invention is a vaccine. It maybe administered directly into the patient, into the affected organ orsystemically i.d., i.m., s.c., i.p. and i.v., or applied ex vivo tocells derived from the patient or a human cell line which aresubsequently administered to the patient, or used in vitro to select asubpopulation of immune cells derived from the patient, which are thenre-administered to the patient. If the nucleic acid is administered tocells in vitro, it may be useful for the cells to be transfected so asto co-express immune-stimulating cytokines, such as interleukin-2 Thepeptide may be substantially pure, or combined with animmune-stimulating adjuvant (see below) or used in combination withimmune-stimulatory cytokines, or be administered with a suitabledelivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and Longenecker et al (1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 CTLs is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 CTLthe fusion partner or sections of a hybrid molecule suitably provideepitopes which stimulate CD4-positive T cells. CD4- and CD8-stimulatingepitopes are well known in the art and include those identified in thepresent invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth in SEQ ID NO:1 or 20 and at least oneadditional peptide, preferably two to 50, more preferably two to 25,even more preferably two to 15 and most preferably two, three, four,five, six, seven, eight, nine, ten, eleven, twelve or thirteen peptides.The peptide(s) may be derived from one or more specific TAAs and maybind to MHC class I and/or class II molecules.

The polynucleotide may be substantially pure, or contained in a suitablevector or delivery system. The nucleic acid may be DNA, cDNA, PNA, CNA,RNA or a combination thereof. Methods for designing and introducing sucha nucleic acid are well known in the art. An overview is provided bye.g. Pascolo S. 2006; Stan R. 2006, or A Mandavi 2006. Polynucleotidevaccines are easy to prepare, but the mode of action of these vectors ininducing an immune response is not fully understood. Suitable vectorsand delivery systems include viral DNA and/or RNA, such as systems basedon adenovirus, vaccinia virus, retroviruses, herpes virus,adeno-associated virus or hybrids containing elements of more than onevirus. Non-viral delivery systems include cationic lipids and cationicpolymers and are well known in the art of DNA delivery. Physicaldelivery, such as via a “gene-gun,” may also be used. The peptide orpeptides encoded by the nucleic acid may be a fusion protein, forexample with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CTLs and helper-T(T_(H)) cells to an antigen, and would thus be considered useful in themedicament of the present invention. Suitable adjuvants include, but arenot limited to, 1018 ISS, aluminum salts, Amplivax, AS15, BCG,CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligands derived fromflagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod (ALDARA),resimiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13, IL-21,Interferon-alpha or -beta, or pegylated derivatives thereof, IS Patch,ISS, ISCOMATRIX, ISCOMs, JuvImmune, LipoVac, MALP2, MF59, monophosphoryllipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V,Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432,OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system, PLG anddextran microparticles, resiquimod, SRL172, Virosomes and otherVirus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys,Aquila's QS21 stimulon, which is derived from saponin, mycobacterialextracts and synthetic bacterial cell wall mimics, and other proprietaryadjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such asFreund's or GM-CSF are preferred. Several immunological adjuvants (e.g.,MF59) specific for dendritic cells and their preparation have beendescribed previously (Dupuis M et al 1998; Allison 1998). Also cytokinesmay be used. Several cytokines have been directly linked to influencingdendritic cell migration to lymphoid tissues (e.g., TNF-α), acceleratingthe maturation of dendritic cells into efficient antigen-presentingcells for T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No.5,849,589, specifically incorporated herein by reference in itsentirety) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23,IL-7, IFN-alpha. IFN-beta) (Gabrilovich et al 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of T_(H1) cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The T_(H1) biasinduced by TLR9 stimulation is maintained even in the presence ofvaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA)that normally promote a T_(H2) bias. CpG oligonucleotides show evengreater adjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg et al 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and AmpliGen, non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4 and SC58175, which mayact therapeutically and/or as an adjuvant. The amounts andconcentrations of adjuvants and additives useful in the context of thepresent invention can readily be determined by the skilled artisanwithout undue experimentation. Preferred adjuvants are dSLIM,Interferon-alpha, -beta, CpG7909, IC31, Imiquimod, resimiquimod,PeviTer, RNA, tadalafil, temozolomide, and JuvImmune.

Preferred adjuvants are dSLIM, BCG, OK432, imiquimod, resimiquimod,GMCSF, interferon-alpha, PeviTer and JuvImmune or combinations thereof.

In a preferred embodiment the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), imiquimod, resiquimod, andinterferon-alpha.

In a still further preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant is imiquimod orresimiquimod. In an even more preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant is the combinationof GM-CSF and imiquimod.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavours, lubricants, etc.The peptides can also be administered together with immune stimulatingsubstances, such as cytokines Δn extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients, 3. Ed. 2000, AmericanPharmaceutical Association and pharmaceutical press. The composition canbe used for a prevention, prophylaxis and/or therapy of adenomatous orcancerous diseases.

The present invention provides a medicament that useful in treatingcancer, in particular glioma and brain cancer, breast cancer, prostatecancer, esophagus cancer, colorectal cancer, renal cancer, pancreaticcancer, squamous cell carcinomas and keratinocytic neoplasms of theskin, leukemia, lung cancer, ovarian cancer, and melanoma.

The present invention includes a kit comprising:

(a) a container that contains a pharmaceutical composition as describedabove, in solution or in lyophilized form;(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, a anti-angiogenesis agent orinhibitor, a apoptosis-inducing agent or a chelator) or a pharmaceuticalcomposition thereof. The components of the kit may be pre-complexed oreach component may be in a separate distinct container prior toadministration to a patient. The components of the kit may be providedin one or more liquid solutions, preferably, an aqueous solution, morepreferably, a sterile aqueous solution. The components of the kit mayalso be provided as solids, which may be converted into liquids byaddition of suitable solvents, which are preferably provided in anotherdistinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably the administration is s.c., and most preferably,i.d. Administration may be by infusion pump.

Since the peptides of the invention derived from BCA, CLIP2, DTNA,NLGNAX, NR2E1, NRCAM and PDPN were isolated from glioblastoma, themedicament of the invention is preferably used to treat glioblastoma.

The present invention will now be described in the following examplesand Figures that describe preferred embodiments thereof, nevertheless,without being limited thereto. For the purposes of the presentinvention, all references as cited herein are incorporated by referencein their entireties.

FIG. 1: Exemplary mass spectrum from IGF2BP3-001 demonstrating itspresentation on primary tumor sample GB6010. NanoESI-LCMS was performedon a peptide pool eluted from the GBM sample GB6010. The masschromatogram for m/z 536.3238±0.001 Da, z=2 shows a peptide peak at theretention time 49.89 min. B) The detected peak in the mass chromatogramat 48.76 min revealed a signal of m/z 536.3239 in the MS spectrum. C) Acollisionally induced decay mass spectrum from the selected precursorm/z 536.3239 recorded in the nanoESI-LCMS experiment at the givenretention time confirmed the presence of IGF2BP3-001 in the GB6010 tumorsample. D) The fragmentation pattern of the synthetic IGF2BP3-001reference peptide was recorded and compared to the generated naturalTUMAP fragmentation pattern shown in C for sequence verification.

FIGS. 2A and 2B show the expression profiles of mRNA of selectedproteins in normal tissues and in 19 glioblastoma samples.

FIGS. 2C and 2D show the expression profiles of mRNA of selectedproteins in normal tissues and in 19 glioblastoma samples

FIG. 3 shows the exemplary in vitro immunogenicity of IMA950 class ITUMAPs

FIG. 4 shows the exemplary binding affinities of HLA class I peptides ofthe invention to A*02

SEQ ID Nos 1 to 24 show the sequences of preferred tumor associatedpeptides according to the present invention.

EXAMPLES

The peptides FTELTLGEF (HLA-Al; PolyPeptide Laboratories, Wolfenbüttel,Germany), LMLGEFLKL (HLA-A2; Clinalfa, Sissach, Switzerland), andEPDLAQCFY (HLA-B35; PolyPeptide Laboratories) were all obtained inpharmaceutical quality.

Example 1 Identification of Tumor Associated Peptides Presented on CellSurface Tissue Samples

Patients' tumor tissues were provided by Höpital Cantonal Universitairede Genève (Medical Oncology Laboratory of Tumor Immunology) andNeurochirurgische Universitäts-Klinik Heidelberg (MolekularbiologischesLabor). Written informed consents of all patients had been given beforesurgery. Tissues were shock-frozen in liquid nitrogen immediately aftersurgery and stored until isolation of TUMAPs at −80° C.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk, K. et al 1991; Seeger, F. H. et al. T 1999) using theHLA-A*02-specific antibody BB7.2 or the HLA-A, -B, -C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Methods:

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (Acquity UPLC system,Waters) and the eluting peptides were analyzed in an LTQ-Orbitrap hybridmass spectrometer (ThermoElectron) equipped with an ESI source. Peptidepools were loaded directly onto the analytical fused-silicamicro-capillary column (75 μm i.d.×250 mm) packed with 1.7 μm C18reversed-phase material (Waters) applying a flow rate of 400 nL perminute. Subsequently, the peptides were separated using a two-step 180minute-binary gradient from 10% to 33% B at flow rates of 300 nL perminute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe micro-ESI source. The LTQ-Orbitrap mass spectrometer was operated inthe data-dependent mode using a TOPS strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30 000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide. FIG. 1 shows an exemplary spectrum obtained fromtumor tissue for the MHC class I associated peptide IGF2BP3-001 and itselution profile on the UPLC system.

Example 2 Expression Profiling of Genes Encoding the Peptides of theInvention

Not all peptides identified as being presented on the surface of tumorcells by MHC molecules are suitable for immunotherapy, because themajority of these peptides are derived from normal cellular proteinsexpressed by many cell types. Only few of these peptides aretumor-associated and likely able to induce T cells with a highspecificity of recognition for the tumor from which they were derived.In order to identify such peptides and minimize the risk forautoimmunity induced by vaccination the inventors focused on thosepeptides that are derived from proteins that are over-expressed on tumorcells compared to the majority of normal tissues.

The ideal peptide will be derived from a protein that is unique to thetumor and not present in any other tissue. To identify peptides that arederived from genes with an expression profile similar to the ideal onethe identified peptides were assigned to the proteins and genes,respectively, from which they were derived and expression profiles ofthese genes were generated.

RNA Sources and Preparation

Surgically removed tissue specimens were provided by two differentclinical sites (see Example 1) after written informed consent had beenobtained from each patient. Tumor tissue specimens were snap-frozen inliquid nitrogen immediately after surgery and later homogenized withmortar and pestle under liquid nitrogen. Total RNA was prepared fromthese samples using TRIzol (Invitrogen, Karlsruhe, Germany) followed bya cleanup with RNeasy (QIAGEN, Hilden, Germany); both methods wereperformed according to the manufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed such that RNA fromeach individual was equally weighted. Leukocytes were isolated fromblood samples of 4 healthy volunteers.

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

Microarray Experiments

Gene expression analysis of all tumor and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual. Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analyzed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalisation, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal kidney sample was arbitrarily setto 1.0.

The expression profiles of the source genes of the present inventionthat highly over-expressed in glioblastoma of the present invention areshown in FIG. 2.

Example 3 In Vitro Immunogenicity for IMA950 MHC Class I PresentedPeptides

In order to obtain get information regarding the immunogenicity of theTUMAPs of the present invention, we performed investigations using awell established in vitro stimulation platform already described by(Walter, S, Herrgen, L, Schoor, O, Jung, G, Wernet, D, Buhring, H J,Rammensee, H G, and Stevanovic, S; 2003, Cutting edge: predeterminedavidity of human CD8 T cells expanded on calibrated MHC/anti-CD28-coatedmicrospheres, J. Immunol., 171, 4974-4978). This way we could showconsiderably high immunogenicity for 13 HLA-A*0201 restricted TUMAPs ofthe invention (in >=50% of tested donors TUMAP-specific CTLs could bedetected) demonstrating that these peptides are T-cell eptiopes againstwhich CD8+ precursor T cells exist in humans (Table 3).

In Vitro Priming of CD8+ T Cells

To perform in vitro stimulations by artificial antigen presenting cells(aAPC) loaded with peptide-MHC complex (pMHC) and anti-CD28 antibody,first we isolated PBMCs (peripheral blood mononuclear cells) from freshHLA-A*02+ buffy coats by using standard density gradient separationmedium (PAA, Cölbe, Germany). Buffy coats were either obtained from theBlood Bank Tübingen or from the Katharinenhospital Stuttgart. IsolatedPBMCs were incubated overnight in T-cell medium (TCM) for human in vitropriming consisting of RPMI-Glutamax (Invitrogen, Karlsruhe, Germany)supplemented with 10% heat inactivated human AB serum (PAA, Cölbe,Germany), 100 U/ml Penicillin/100 μg/ml Streptomycin (Cambrex, Verviers,Belgium), 1 mM sodium pyruvate (CC Pro, Neustadt, Germany) and 20 μg/mlGentamycin (Cambrex). CD8+ lymphocytes were isolated using the CD8+MACSpositive selection kit (Miltenyi, Bergisch Gladbach, Germany) accordingto the manufacturer's instructions. Obtained CD8+ T-cells were incubateduntil use in TCM supplemented with 2.5 ng/ml IL-7 (PromoCell,Heidelberg, Germany) and 10 U/ml IL-2 (Chiron, Munich, Gemany).Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed as described before (Walter et al., 2003) withminor modifications. Briefly, biotinylated recombinant HLA-A*0201molecules lacking the transmembrane domain and biotinylated at thecarboxy terminus of the heavy chain were produced following a methoddescribed by (Altman et al., 1996). The purified costimulatory mouseIgG2a anti human CD28 Ab 9.3 (Jung et al., 1987) was chemicallybiotinylated using Sulfo-N-hydroxysuccinimidobiotin as recommended bythe manufacturer (Perbio, Bonn, Germany). Beads used were 5.60 μm largestreptavidin coated polystyrene particles (Bangs Laboratories,Illinois/USA). pMHC used as positive and negative controls wereA*0201/MLA-001 (peptide ELAGIGILTV from modified Melan-A/MART-1) andA*0201/DDX5-001 (YLLPAIVHI from DDX5) or A*0201/HBV-001 (FLPSDFFPSV),respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of600 ng biotin anti-CD28 plus 200 ng relevant biotin-pMHC (high densitybeads) or 2 ng relevant plus 200 ng irrelevant (pMHC library) MHC (lowdensity beads). Stimulations were initiated in 96-well plates byco-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washed coated beads in 200μl TCM supplemented with 5 ng/ml IL-12 (PromoCell) for 3-4 days at 37°C. Half of the medium was then exchanged by fresh TCM supplemented with80 U/ml IL-2 and incubating was continued for 3-4 days at 37° C. Thisstimulation cycle was performed for a total of three times. Finally,tetrameric analyses were performed with fluorescent MHC tetramers(produced as described by (Altman et al., 1996)) plus antibody CD8-FITCclone SK1 (BD, Heidelberg, Germany) on a four-color FACSCalibur (BD).Peptide specific cells were calculated as percentage of total CD8+ Tcells. Evaluation of tetrameric analysis was done using the software FCSExpress (De Novo Software). In vitro priming of specific tetramer+ CD8+lymphocytes was detected by appropriate gating and by comparing tonegative control stimulations. Immunogenicity for a given antigen wasdetected if at least one evaluable in vitro stimulated well of onehealthy donor was found to contain a specific CD8+ T-cell line after invitro stimulation (i.e. this well contained at least 1% of specifictetramer+ among CD8+ T-cells and the percentage of specific tetramer+cells was at least 10× the median of the negative control stimulations).

In Vitro Immunogenicity for IMA950 Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific tetramer staining for twopeptides of the invention are shown in FIG. 3. Results for 13 peptidesfrom the invention are summarized in Table 3.

TABLE 3 In vitro immunogenicity of highly immunogenic HLA class Ipeptides of the invention Positive donor/ Positive wells/ Antigen donorstested wells tested BCA-001  60%  5% BCA-002  75% 35% CLIP2-001  75%  6%CSP-001 100% 57% FABP7-001 100% 27% IGF2BP3-001  50% 21% NES-001  75%38% NLGN4X-001 100% 62% NRCAM-001  86% 39% PDPN-001  60% 11% SLCO1C1-001 60%  7% TNC-001  60% 30% TNC-002  50% 14%

In addition to these results obtained from healthy blood donors, thepeptides BCA-002, CHI3L1-001, and NLGN4X-001 were also tested in a smallnumber of glioblastoma patients. All peptides proved to be immunogenicto a similar extent compared with healthy donors, demonstrating theexistence of precursor T cells in a relevant target population for thevaccine.

Example 4 Binding of HLA Class I-Restricted Peptides of the Invention toHLA-A*0201 Objective and Summary

The objective of this analysis was to evaluate the affinity of the HLAclass I peptides to the MHC molecule coded by the HLA-A*0201 allele asthis is an important parameter for the mode of action of peptides aspart of cancer immunotherapies. Affinities to HLA-A*0201 were medium tohigh for all tested HLA class I-restricted peptide 0 of the invention,with dissociation constants in the range of the positive control peptideHBV-001, a known strong A*02 binder derived from hepatitis B virus coreantigen. These results confirmed the strong binding affinity of alltested HLA class I peptides of the present invention.

Principle of Test

Stable HLA/peptide complexes consist of three molecules: HLA heavychain, beta-2 microglobulin (b2m) and the peptidic ligand. The activityof denatured recombinant HLA-A*0201 heavy chain molecules alone can bepreserved making them functional equivalents of “empty HLA-A*0201molecules”. When diluted into aqueous buffer containing b2m and anappropriate peptide, these molecules fold rapidly and efficiently in anentirely peptide-dependent manner. The availability of these moleculesis used in an ELISA-based assay to measure the affinity of interactionbetween peptide and HLA class I molecule (Sylvester-Hvid et al., 2002).

Purified recombinant HLA-A*0201 molecules were incubated together withb2m and graded doses of the peptide of interest. The amount of denovo-folded HLA/peptide complexes was determined by a quantitativeELISA. Dissociation constants (KD values) were calculated using astandard curve recorded from dilutions of a calibrant HLA/peptidecomplex.

Results

Results are shown in FIG. 4. A lower KD value reflects higher affinityto HLA-A*0201. All tested peptides of the invention had a strongaffinities to HLA-A*0201 around the KD for the positive control peptideHBV-001, a known strong A*02 binder. Thereby, all class I TUMAPs of theinvention have a strong binding affinity to the MHC molecule A*02.

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1. A peptide comprising a sequence that is selected from SEQ ID NO:1 toSEQ ID NO:30, and/or a first variant thereof that is at least 85%homologous to SEQ ID NO:1 to SEQ ID NO:30, and/or a second variantthereof that induces T cells cross-reacting with said first variantpeptide, wherein said peptide is not an underlying full-lengthpolypeptide.
 2. The peptide and/or variant thereof according to claim 1,wherein said peptide or variant thereof maintains an ability to bind toa molecule of the human major histocompatibility complex (MHC) class-Ior -II, and wherein said peptide is capable of stimulating CD4 and/orCD8 T cells.
 3. The peptide and/or variant according to claim 1 whereinan amino acid sequence thereof comprises a continuous stretch of aminoacids from SEQ ID NO:1 to SEQ ID NO:30.
 4. The peptide and/or variantaccording to claim 1, wherein said peptide is selected from a peptidehaving a specific HLA-subtype according to table 2, and is capable ofstimulating CD8 cells, and wherein said peptide comprises a specificanchor amino acid-motif as depicted in table
 2. 5. The peptide and/orvariant according to claim 1, wherein said peptide or variant thereofhas an overall length of between 8 and 100 amino acids.
 6. The peptideand/or variant according to claim 1, wherein said peptide is modifiedand/or includes non-peptide bonds.
 7. The peptide and/or variantaccording to claim 2, wherein said peptide is a fusion protein,optionally comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii).
 8. A nucleic acid encoding apeptide and/or variant of claim
 1. 9. The nucleic acid of claim 8,further comprising an expression vector, wherein the expression vectorcomprising the nucleic acid is capable of expressing the peptide and/orvariant encoded by the nucleic acid.
 10. A method of treating neuronaltumors, the method comprising administering a peptide and/or variant ofclaim
 1. 11. A host cell comprising a nucleic acid of claim 8, andwherein said host cell is an antigen presenting cell.
 12. A method ofproducing a peptide and/or variant of claim 1, the method comprising:culturing a host cell comprising a nucleic acid encoding said peptideand/or variant; or culturing a host cell comprising an expression vectorcomprising a nucleic acid encoding said peptide and/or variant; andisolating the peptide from the host cell or from a culture medium. 13.An method for producing activated cytotoxic T lymphocytes (CTL), themethod comprising contacting in vitro CTL with antigen-loaded humanclass I or II MHC molecules expressed on the surface of a suitableantigen-presenting cell or an artificial construct mimicking anantigen-presenting cell for a period of time sufficient to activate saidCTL in an antigen specific manner, wherein said antigen is a peptideand/or variant of claim 1
 14. An activated cytotoxic T lymphocyte (CTL)produced according to claim
 13. 15. A method for killing target cells ina patient, in which said target cells aberrantly express a polypeptidecomprising an amino acid the method comprising administering to thepatient an effective number of activated cytotoxic T lymphocytes (CTL)of claim
 14. 16. A method of manufacturing a cancer vaccine, said methodcomprising using a peptide and/or variant of claim 1 and/or a nucleicacid encoding said peptide and/or variant and/or a vector comprisingsaid nucleic acid.
 17. The method according to claim 16, wherein saidcancer is selected from the group consisting of astrocytoma, pilocyticastrocytoma, dysembryoplastic neuroepithelial tumor, oligodendrogliomas,ependymoma, glioblastoma multiforme, mixed gliomas, oligoastrocytomas,medulloblastoma, retinoblastoma, neuroblastoma, germinoma, teratoma,gangliogliomas, gangliocytoma, central gangliocytoma, primitiveneuroectodermal tumors (PNET, e.g. medulloblastoma, medulloepithelioma,neuroblastoma, retinoblastoma, ependymoblastoma), tumors of the pinealparenchyma (e.g. pineocytoma, pineoblastoma), ependymal cell tumors,choroid plexus tumors, neuroepithelial tumors of uncertain origin (e.g.gliomatosis cerebri, astroblastoma), glioblastoma prostate tumor, breastcancer, esophageal cancer, colorectal cancer, clear cell renal cellcarcinoma, lung cancer, CNS, ovarian, melanoma pancreatic cancer,squamous cell carcinoma, leukemia medulloblastoma, colon, rectum,stomach, kidney, lung, pancreas, prostate, skin, other tumors which showan overexpression of survivin and/or CSPG4, and other proteins fromwhich the peptides of SEQ ID NO:1 to SEQ ID NO:30 are derived.
 18. A kitcomprising: (a) a container that contains a pharmaceutical compositioncontaining, in solution and/or in lyophilized form: (i) a peptide and/orvariant of claim 1, (ii) a nucleic acid encoding said peptide and/orvariant; (iii) an expression vector comprising said nucleic acid; (iv) acell comprising said nucleic acid and/or said expression vector; or (v)an activated cytotoxic T lymphocyte according produced using saidpeptide and/or variant. (b) optionally, a second container comprising adiluent and/or reconstituting solution; (c) optionally, at least onefurther peptide selected from the peptides according to SEQ ID NOs 1 to30, and (d) optionally, instructions for (i) use of the diluent and/or(ii) reconstitution and/or use of a lyophilized formulation.
 19. The kitaccording to claim 18, further comprising one or more of (e) a buffer,(f) a diluent, (g) a filter, (h) a needle, and/or (i) a syringe.
 20. Amethod for distinguishing glioblastoma from other forms of cancercomprising analyzing the expression of BCA, CLIP2, DTNA, NLGNAX, NR2E1,NRCAM and/or PDPN in a sample obtained from the brain or anothertumorous specimen from a subject to be diagnosed.
 21. A kit formeasuring expression level of BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAMand/or PDPN as (a) glioblastoma marker gene(s), comprising at least oneantibody that specifically binds a chosen glioblastoma markerpolypeptide, and/or at least one nucleic acid that specificallyhybridizes with BCA, CLIP2, DTNA, NLGNAX, NR2E1, NRCAM and/or PDPN mRNA,and, optionally, a control, optionally primary and/or secondaryantibodies when appropriate, and optionally a further reagent which isoptionally a detectable moiety, an enzyme substrate, and/or a colourreagent.